Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.For intracellular  Chlamydiaceae are a global health problem. The Gram-negative obligate intracellular pathogens depend on eukaryotic host cells to maintain their unique biphasic developmental cycle. One elusive phenomenon of the chlamydial biology has fascinated researches for two decades: for endobacteria, such as Chlamydiaceae, there is no need to resist osmotic challenges and a functional cell wall has not been detected in these pathogens so far  and show resistance to azithromycin  is essential for the incorporation of cell wall building blocks at the septal cell wall and for cell division.The bacterial cell wall consists of peptidoglycan, a polymer of long chains with alternating sugar units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked via flexible peptide bridges. Peptidoglycan is found in all eubacteria with the exception of some obligate intracellular species. Biosynthesis of peptidoglycan takes place in three stages Figure . In the C. pneumoniae and subsequently mediate activation of the transcription factor NFkB which plays a key role in regulating the immune response to infection  and FtsK, and possess, despite their spherical shape, the cytoskeletal protein MreB  cofactor requiring alanine racemases Alr and DadX. Therefore, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae have remained unclear for years.We proposed that retaining biosynthesis of lipid II in cell wall-lacking “minimal bacteria,” like e Figure . A recenin vitro for GlyA from E. coli  shows an effect on the D-Ala auxotrophic phenotype of an E. coli Δ alrΔdadX racemase double mutant strain. Our experiments in liquid and solid culture revealed that chlamydial GlyA did not completely reverse the need of exogenous D-Ala of the racemase mutant strain but favored its growth under D-Ala limited conditions  and colorimetrically quantified. The chlamydial GlyA converted L-Ala to D-Ala in vitro exhibiting weak racemase activity in comparision to the enzyme from B. stearothermophilus  was annotated  agar plates containing 30 μg/ml chloramphenicol and 100 μg/ml ampicillin, respectively. The temperature sensitive E. coli Δ alrΔ dadX racemase double mutant TKL-10 was maintained on LB agar plates containing 25 μg/ml thymine.E. coli TKL-10 Δ alrΔ dadX was transformed with pET21b-glyACp and grown in liquid or on solid LB medium supplemented with 5 mg/L D-Ala, 50 μM PLP, 25 μg/ml thymine, 50 μg/ml ampicillin and 0.1 mM IPTG at 42°C. For each experiment three independently generated transformants were used and controls with E. coli TKL-10 Δ alrΔ dadX harboring the empty pET21b vector were carried out.glyA gene from C. pneumoniae GiD was amplified by PCR using primer glyACp_f (5′-ATGGTAGGTCTCAGGCCTTGCTAAAAGTTTTTGAGAAATTTAAGA-3′) and glyACp_r (5′-ATGGTAGGTCTCAGCGCTAACTAAAGCTTCTAAATCAATTTCAGG-3′) and cloned into pASK-IBA2c  using the BsaI restriction site to generate an N-terminal OmpA-leader peptide fused, C-terminal Strep-tagged protein for periplasmic overproduction. For cytoplasmic expression in complementation assays, glyA was amplified with primers glyACp_pET21_f (5′-CGTCTTTAGAAGCATATGCTAAAAG-3′) and glyACp_pET21_r (5′-GTCTCTGCGGCCGCAACTAAAGCTTC-3′) and cloned into pET21b  using NdeI and NotI restriction sites.The E. coli JM83 cells, transformed with pASK-IBA2c-glyACp, were grown in no salt LB in presence of 30 μg/ml of chloramphenicol, 250 mM sucrose and 50 mM L-serine at 30°C. After induction at an OD600 of 1.2 with 200 ng/ml anhydrotetracyline (AHT), 50 μM PLP and 200 μM of folinic acid were added and the cells were incubated for 4 h at 25°C. The purification of GlyACp was performed using the protocol for cleared lysates recommended by the manufacturer  with small modifications: the buffers contained 2% N-lauroylsarcosine (or 0.1% N-lauroylsarcosine in the washing and elution buffer), 2 mM 1,4-dithiothreitol (DTT) and 50 μM PLP. Purity of the protein was controlled using SDS-PAGE.Cp was determined in a DAAO coupled enzymatic assay system as described previously with slight modifications  as described before  was used as positive control. PLP containing enzymes have been described to show weak transamination activity converting alanine to pyruvate. As a control for potential L-Ala transamination activity of GlyACp, we ran experiments in the absence of DAAO. No GlyACp catalyzed production of pyruvate was detected. For GlyACp inhibition assays, 10 mM D-cycloserine was added and both enzymatic steps were carried out consecutively with a step of heat deactivation in between as DAAO is sensitive to D-cycloserine inhibition.Racemase activity of GlyAThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes  D. melanogaster and D. simulans, repress transposable elements and satellite DNAs. These proteins also help regulate the length of the atypical Drosophila telomeres, which are themselves made of domesticated transposable elements. Our data suggest that these proteins are part of the adaptive machinery that allows the host to respond to changes and increases in heterochromatin and to maintain the activity of genes located within or adjacent to heterochromatin.Sister species capable of mating often produce hybrids that are sterile or die during development. This reproductive isolation is caused by incompatibilities between the two sister species' genomes. Some hybrid incompatibilities involve genes that encode rapidly evolving proteins that localize to heterochromatin. Heterochromatin is largely made up of highly repetitive transposable elements and satellite DNAs. It has been hypothesized that rapid changes in heterochromatic DNA drives the changes in these HI genes and thus the evolution of reproductive isolation. In support of this model, we show that two rapidly evolving HI proteins, Lhr and Hmr, which reproductively isolate the fruit fly sister species  As populations diverge, their ability to reproduce with each other diminishes. Hybrid incompatibility (HI), the reduced viability and fertility of interspecific hybrids, is a major cause of reproductive isolation between nascent species and thus an important contributor to speciation. Many of the genes causing HI show evidence of adaptive evolution, typically manifest as excessive numbers of amino-acid-changing mutations compared to neutral expectations Adaptive evolution therefore does ultimately lead to HI, but if we wish to identify the evolutionary forces that drive the divergence of HI genes, then we need to understand the function of these genes within species. The mechanisms by which HI genes cause sterility or lethality are important but separate issues. In fact, it remains uncertain whether the wild type functions of HI genes are generally predictive of the deleterious phenotypes that they cause within hybrids.Hybrid male rescue (Hmr) gene causes large reductions in hybrid fitness D. melanogaster, however, have only moderate effects on fertility and provide few insights into mechanistic underpinnings D. melanogaster, but the genes have many pleiotropic functions and the challenge is to pinpoint which one(s) are driving evolutionary divergence.Pinpointing the function of HI genes and the causes of their adaptive evolution is a challenging goal. For example, the Lethal hybrid rescue (Lhr) and Hmr, which interact to cause F1 hybrid male lethality between D. melanogaster and D. simulansHmr this sequence divergence appears to be required for hybrid lethality because the D. melanogaster ortholog of Hmr causes hybrid lethality but the D. simulans ortholog does not Lhr, however, both orthologs have hybrid lethal activity, with D. simulans Lhr having greater activity due to its higher expression level in hybrids Lhr coding sequence divergence makes some contribution to hybrid lethality. Furthermore we found that Lhr from the more diverged species D. virilis has no hybrid lethal activity, suggesting that more extensive coding sequence divergence does have substantial functional consequences Here we investigate two hybrid lethality genes, Hmr and Lhr function within each of the hybridizing species. Loss of function alleles of Hmr and Lhr are strong suppressors of hybrid lethality, but are largely viable within D. melanogaster and D. simulans, respectively These previous studies leave unanswered the fundamental question of what evolutionary force is driving adaptive sequence change, and necessitate a detailed understanding of Lhr  protein localizes to heterochromatin D. simulans and D. melanogaster have great potential for addressing this question because they differ substantially from each other in genome size Hmr and Lhr are required to repress transcription from both TEs and satellite DNAs. Hmr and Lhr also regulate telomeres, a third specialized type of heterochromatic sequence that serves to protect the ends of linear chromosomes D. simulans mutation in Lhr, comparative cytology, and interspecific complementation with Hmr transgenes to identify classes of TEs and satellites that are regulated differentially between the species. We conclude that Hmr and Lhr provide an adaptive defense against multiple classes of repetitive DNA sequences that change rapidly in evolutionary time, can reduce host fitness, and have high potential to provoke genetic conflict.Genetic conflicts with selfish DNAs have been proposed as an important driver of HI Hmr transgene  studies from embryo extracts and found that mel-Lhr-HA and mel-Hmr-FLAG co-IP . mel-Lhre levels , demonste levels .Hmr and Lhr from D. melanogaster interact, suggesting that the co-IP reflects a direct interaction between the proteins . However, comparison of KOLhr with a background-matched +Lhr control  transcripts. Using two different TE mapping methods (see HeT-A (350.7 fold) and TART (51.76 fold), the LTR retrotransposon copia (19.8 fold), and the DNA transposon bari-1 (44.7 fold). TEs expressed only in the somatic follicle cells, such as Gypsy (3.8 fold) and Zam (7 fold) were also upregulated. In addition, qRT-PCR in two different genetic backgrounds confirmed the massive increase in HeT-A transcript levels Hmr, abbreviated below as −Hmr). We compared it to a heterozygous control (−/y w Hmr+Df(1)Hmr, abbreviated below as −Hmr/+Hmr) because it closely matches the genetic background of the mutant genotype, and also serves as a control for Hmr transgenic genotypes that are described below. We found that 55 different TE families are upregulated at least 2 fold in Hmr mutants, with 14 being upregulated at least 10 fold Hmr and +/Y D. melanogasterHmr male larvae. This also served as a control for experiments described below to address whether TE mis-expression may be contributing to hybrid lethality. We found that 31 TEs exhibit a statistically significant ≥2 fold upregulation  show a statistically significant misregulation in either Lhr or Hmr mutants  KOLhr  LhrKO . We thenr piRNAs . This pand LhrKO . We alsoKO  repeats and are therefore not expected to contribute to TE repression; their misregulation instead suggests that Lhr is required for regulating chromatin states at telomeres.We searched further for possible defects in piRNA production by examining piRNAs that map to 122 primary-piRNA-generating heterochromatic clusters in LhrKO . Some ofHeT-A, are not significantly different between KOLhr and +Lhr, suggesting that Lhr is not generally required for siRNA biogenesis  RNA-Seq data. Unlike in KOD. melanogaster Lhr, we found few satellite reads in either genotype and no significant differences between them. We conclude that Lhr has a unique role in D. melanogaster to repress satellite DNA transcription. The AACAC satellite that Lhr co-localizes with in D. melanogaster Hmr), and performed RNA-Seq on ovarian mRNA. Our expectation was that divergence of Hmr between the orthologs might manifest as the failure of sim-Hmr-FLAG to complement the derepression of TEs in −Hmr.To examine potential differences in TE and satellite regulation, we used parallel −Hmr/+Hmr to −; ø{mel-Hmr-FLAG}/+Hmr, as each genotype has one wild type copy of +Hmr. The majority of the upregulated TEs in −Hmr . The differential expression of these 5 families likely reflects the inability of mel-Hmr-FLAG to fully complement −Hmr and the higher expression level of sim-Hmr-FLAG.We did not find any difference in satellite DNA expression; however, we found 11 TE families that are differentially expressed between the transgenic genotypes . Five ar−; ø{sim-Hmr-FLAG}/+Hmr than in −; ø{mel-Hmr-FLAG}/+Hmr, implying that sim-Hmr-FLAG is unable to fully complement the derepression of these elements. BS and Doc6  elements are present at a mean frequency of about 0.1 in a population of Portuguese D. melanogasterBS3, Circe, Helena, and FW2) are near 1, suggesting that these TEs are fixed and therefore currently inactive in D. melanogaster. Helena, though, appears to have been active more recently within D. simulans BS, Doc6 and Helena are candidates for future investigation of co-evolution with Hmr in either D. melanogaster or D. simulans.More intriguing are 6 TE families that are 2–6× more highly expressed in Lhr and Hmr are required for TE repression within D. melanogaster and D. simulans, we investigated TE activity in lethal (+Hmr) hybrid male larvae. Because most TEs have different expression levels between D. melanogaster and D. simulans, we defined mis-regulated TEs as being at least two-fold higher than both parental species, as done in a previous analysis +Hmr) hybrid male larvae with 2 others being downregulated  in the activity of 5 TE families in living hybrids. None of these are significantly upregulated in e larvae . They inSu(var)205 which enodes HP1a Hmr and Lhr are viable. Furthermore, Hmr and Lhr do not localize to the 359 bp satellite which forms a substantial fraction of X-linked pericentric heterochromatin We and others previously reported that Lhr  interacts with HP1a Hmr dosage is in concordance with previous studies, such as the previous observation that a ∼9.7 kb +Hmr transgene causes dosage-dependent lethality to hybrid females Lhr dosage The interaction of Hmr and Lhr with HP1a has recently been independently reported Hmr and Lhr mutants versus RNAi knockdown. Thomae et al report a high rate of mitotic defects in Lhr RNAi knockdown tissue culture cells, yet we found that KOLhr flies are almost fully viable Hmr allele, a deletion of the 5′ end of Hmr. Further work is necessary to determine if these discrepancies reflect phenotypes associated with the use of RNA interference or differences between assaying whole animals versus tissue-culture cells, such as the aneuploid state of cultured cell lines One area of possible discrepancy is the viability effects and cellular phenotypes associated with able see , as are Several HIs involve heterochromatin proteins or heterochromatic sequences, leading to the suggestion that genetic conflicts between selfish DNAs and host fitness are an important force that is driving the evolution of HI TE and satellite abundance varies widely among species and is a major contributor to genome-size variation. The evolutionary causes of this variation have been widely debated for many years Hmr and LhrSelectionist models fit better for active repeats, and must be invoked if the adaptive evolution of heterochromatin proteins is proposed to reflect co-evolution with repetitive DNA. One model is that some repeats are co-opted for host functions. Drosophila's telomeric retrotransposons are a relevant example that is discussed below. We also consider three, non-mutually exclusive selective costs associated with repetitive DNA when discussing the evolution of One potential cost arises from the overall load of repetitive DNAs, including increased genome size and instability. A second is direct genetic conflict. We define genetic conflict here to refer to fitness costs imposed by selfish DNAs that have evolved specific mechanisms to increase their transmission TEs define selfish DNA Hmr and Lhr, many piRNA pathway genes are also evolving under positive selection Lhr and Hmr are co-evolving with the piRNA pathway proteins. However, the lack of major perturbations in the piRNA pool in KOLhr suggests that Lhr and Hmr function downstream or independently of piRNA biogenesis. Piwi, guided by piRNA, has been proposed to recruit repressive heterochromatin components including HP1a and histone methyl transferases to transposable elements Like Ago3 is moderately down-regulated in both KOLhr (3.4 fold) and −Hmr (∼2 fold)  , S6, likol LhrKO .Lhr, Hmr and the telomeric TEs resembles symbiosis. These TEs have been domesticated by Drosophila species for tens of millions of years to serve a vital host function, and thus are not considered selfish DNA Hmr and Lhr mutants, in some cases more than 100 fold. We also observed increases in HeT-A DNA copy number in Hmr and Lhr stocks. Increased telomeric TE expression does not necessarily increase HeT-A DNA copy number and cause longer telomeres, suggesting that multiple factors control telomere length Lhr and Hmr must control multiple processes at the telomere. This is supported by the localization of both proteins to the telomere cap, a protective structure that prevents telomere fusions KOLhr of piRNAs from three TAS-repeat containing sub-telomeric piRNA clusters is particularly intriguing. piRNA production from clusters is dependent on them maintaining a heterochromatic state Lhr is required for TAS piRNA expression while it acts as a repressor in most other circumstances.While TE repression is typically viewed in terms of genetic conflicts, the relationship between Hmr and Lhr with satellite DNAs. We found that D. melanogaster Hmr and Lhr proteins localize to and repress transcripts from GA-rich satellites. GA-rich satellites are ∼8 fold less abundant in D. simulansD. erectaD. simulans reflects an increase in D. melanogaster. Similarly we discovered that mel-Lhr-HA localizes to AACAC in D. melanogaster, a repeat that is absent in D. simulansD. melanogaster. Our results suggest that Lhr and Hmr may have evolved in D. melanogaster to mitigate the deleterious consequences of satellite expansion, which can include ectopic recombination, increased genome size, and destabilized chromosome segregation We discovered several striking examples that suggest species-specific co-evolution of D. melanogasterD. melanogaster spn-E mutants, suggesting that RNA interference or piRNA pathways control satellite transcript levels Satellite transcripts have been reported from various tissues in wild type Lhr and Hmr from both D. melanogaster and D. simulans regulate heterochromatic repetitive DNAs but very few genes. This finding is consistent with previous analyses demonstrating that some functions of these genes are conserved between species Lhr and Hmr are rapidly evolving, raising the question of whether specific repetitive DNAs are directly driving the adaptive evolution of the Lhr and Hmr coding sequences between species. A simple prediction is that D. simulans orthologs should fail to fully repress such repeats when placed into D. melanogaster, a prediction that we tested for Hmr.We find that at a broad scale, BS non-LTR retrotransposon is significantly derepressed in −D. melanogaster Hmr and KOLhr, and in 1D. simulans Lhr mutants. Interestingly, BS appears to be transpositionally active in D. melanogaster but inactive in D. simulansBS was active in the common ancestor and regulated by Hmr and Lhr. The genes would continue to co-evolve with BS in D. melanogaster, making the sim-Hmr ortholog less effective at repressing BS elements in D. melanogaster. In this scenario Hmr and Lhr are engaged in a recurrent genetic conflict with BS elements that leads to their sequence divergence. Consistent with this prediction we found significantly higher expression in −; ø{sim-Hmr-FLAG}/+Hmr compared to −; ø{mel-Hmr-FLAG}/+Hmr.The Copia shows a different pattern, with ∼20-fold up-regulation in KOLhr but only ∼2-fold in 1Lhr (and only when mapping to the consensus-sequence database), as well as significant derepression in −Hmr. Copia expression level can be high in D. melanogaster but is variable among populations. In contrast, copia elements in D. simulans typically contain deletions in regulatory elements required for expression, and transcripts are undetectable by Northern blot analysis Hmr and Lhr could be D. melanogaster host factors that defend against a TE that is currently active within the species. However, copia was fully repressed in −; ø{sim-Hmr-FLAG}/+Hmr, demonstrating that adaptive divergence of Hmr by itself does not affect copia regulation.Hmr divergence, including no effects on satellite DNAs  was used to PCR amplify two genomic fragments from y; cn bw sp (D. melanogaster) genomic DNA. The 3768 bp Lhr upstream fragment, including 128 bp of the coding region of Lhr, was amplified with primers LUF-Fwd: ggcgcgccAACAGGGTCGGCTGTCACATTT5′- tt and LUF-Rev: ggcgcgccGCGAGCATCTCCATGAGCAG5′-tt (Tm = 63°C) and cloned into the AscI site of pW25 using the underlined sequences. The 3935 bp Lhr downstream fragment that includes 806 bp of the Lhr coding region was amplified with primers LDF-Fwd: GCGGCCGCAGGTGGAGCCCAAAATGGACG5′-AA and LDF-Rev: GCGGCCGCCACACATTGCGAATGCA G AAA5′- AA (Tm = 65°C) and cloned into the NotI site using the underlined sequences. Restriction digestion was used to pick a clone in which the 2 inserts and the mini-white gene were in the same orientation.We used the pW25 donor vector and ends-out homologous recombination method to make an 1118w (Genetic Services) and a transgenic line, +, Lhr-KO}5-1P{w, with a lethal insertion on the X chromosome was obtained. +, Lhr-KO}5-1/FM6P{w females were crossed to +, hs-flpase}y w; P{ry, + hs-I-Sce}/TM6P{v, Ubx males. Two to three day-old larvae were heat shocked and +, Lhr-KO}5-1/y w P{ry+, hs-flpase}P{w, + hs-I-Sce}P{v/+ female progeny were crossed to 1118w males. Rare w+ sons were screened for homologous recombination events by PCR. Primer pairs Lhr-f15′- TTCGCACGTTGTGTTCAAGTAA-3′, /Lhr-r1 5′-GTAGCTTTCTCTTGGCGCTCTT-3′ and Lhr-f2 5′- AACGTGCTCGTAGCTTTGGT-3′/, Lhr-r2 5′-TCGCGAAAATACTTCCGTCT-3′ (Tm = 58°C) produce no amplicons in the presence of the white insertion. Attempts to remove the +w marker by Cre recombination were unsuccessful and the +w-disrupted Lhr locus was designated as KOLhr.The construct was injected into a strain of D. melanogaster Lhr can weakly rescue D. melanogaster-D. mauritiana hybrid males to the pharate adult stage KOLhr homozygous females to D. mauritiana males at 18°, we obtained 10.6% rescue of live males . The stronger rescue observed here may be due to the fact that the mothers of the cross were homozygous for the KOLhr allele, since Lhr likely has strong maternal expression based on its high protein abundance in early embryos To test the genetic effects of this mutation, we took advantage of a recent observation that a deficiency chromosome which deletes D. melanogaster Hmr-FLAG transgene was made by inserting a 3× FLAG tag sequence Hmr using fusion PCR into plasmid p72, which is a pCaSpeR2 vector containing a ∼9.7 kb fragment of the Hmr region Hmr fragments (L-arm and R-arm) were amplified from p72 with iProof polymerase by using primer pairs 739/738 and 736/740, respectively. The primers 738 and 736 contain sequence encoding the FLAG tag and partially overlap to allow fusion in the subsequent stage. The primers 739 and 740 were combined with L-arm and R-arm products to produce a fused partial fragment of Hmr containing the 3× FLAG sequence. This fragment was cloned into the pCR-BluntII-Topo vector (Invitrogen) and sequenced completely between the AvrII and KpnI restriction sites. The AvrII/KpnI fragment was then cloned into the corresponding sites of the p72 plasmid. A 300 bp fragment containing the attB site was then PCR amplified from plasmid pTA-attB  using primers 502 and 503 and cloned into the NotI site. This fragment was digested with NotI (on the ends of 502 and 503), gel purified, and inserted into the NotI site of the plasmid containing Hmr-FLAG. We refer to this transgene as mel-Hmr-FLAG.A D. melanogaster Hmr-HA transgene was made by inserting a 3XHA epitope tag between codons 466 and 467 of Hmr. Primers 215/1246 and 1247/495 were used to amplify 573 and 316 bp fragments, respectively. Primers 1246 and 1247 overlap and encode the HA tag. Fusion PCR containing these 2 products and primers 215/495 was performed. The PCR product was cloned into pCR-Blunt II-TOPO, and the insert was checked by sequencing. The insert was then cloned using SpeI and BsiWI back into a modified p72 containing an attB site inserted into the NotI site. The orientation and presence of the HA tag were checked by double digests and PCR. We refer to this transgene as mel-Hmr-HA.A D. simulans Hmr-FLAG transgene was made by inserting the 3× FLAG tag sequence upstream of the stop codon in p89, a pBluescript II KS(+) plasmid containing the D. simulans Hmr insert that was used for the p92 transformation construct in HpaI site near one end and a NotI site near the other. The NotI site was destroyed during cloning; however, the pCR-Blunt II-TOPO vector contains a NotI site within 40 bp of the destroyed sequence. The insert was then cloned back into p89 using HpaI and NotI. The orientation of the insert, as well as the addition of the FLAG tag, was checked by double digest with ClaI and HpaI. The D. simulans Hmr-FLAG insert was then removed as a SacII fragment. Klenow enzyme was used to fill-in the ends to allow cloning into the StuI site of pCaSpeR2 containing an attB site inserted at its NotI site. We refer to this transgene as sim-Hmr-FLAG.A D. simulans Hmr-HA transgene was made from plasmid p89 by inserting the HA tag at the region orthologous to mel-Hmr-HASpeI and BlpI. Blunt end ligation, used for sim-Hmr-FLAG above, proved inefficient for transferring the insert into the transformation vector. Therefore an XbaI site was added to the 3′ end of Hmr-HA by amplifying the entire insert using primers 1402/1403. The PCR product was then gel purified and cloned back into pCR-Blunt II-TOPO. The polylinker contains an XbaI site 5′ to the insert, allowing us to clone the entire insert into the XbaI site of pCaSpeR2 containing an attB site inserted at its NotI site. We refer to this transgene as sim-Hmr-HA.The Hmr transgenes . 739: AGCCAAATTGCCGACAGTAGCCAAG; 738: ATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCAGGCGGTGGCGGATTGACCTTG; 736: GACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAGCTCTCGAAACTTTTGGCACACGTAG; 740: TTGTACTGCCATTAGGTATAGCTAACCATCC; 502: AAACCCGCGGCCGCATGCCCGCCGTGACCGTC; 503: AAACCCGCGGCCGCGATGTAGGTCACGGTCTCG; 152: TCTTCTTAGACTGCGGGTTG; 215: CAGCGCATGCGCGGCACCGTAT; 1246: ATAGTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAACATCGTATGGGTACATTGCACTGTTGGTCATGCTCGT; 1247: TCCCTATGACGTCCCGGACTATGCAGGATCCTATCCATATGACGTTCCAGATTAC;GCTAGCACTGCCACAAGCATTGG; 495: GACACGCCCGTTCCCATAGT; 751: ACAGCGATTTGCGCAAGCCG; 753: TCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCAGGCGGTGGCGGATTTGCCTTCTTGGCGTATTTAGA; 750: GTGAATTGTAATACGACTCACTATAGGGCG; 752: GACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAGCTCTCGAATCATTGGCACACG; 135: GAGGAGGACCCCACCTATAACTAC; 1365: ATAGTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAACATCGTATGGGTATGCACTGTTAGAAATGCTTGTGCTG; 1364: GCTGGCAATTTGGACTTTGT; 1402: GCGGGCGGTCATTATTAA; 1403: TATCTAGAGCGGCCGCGAGCTCTAATA.Oligonucleotides for P{CaryP}attP2 integration site at cytological position 68A4 D. melanogaster transformants were recognized by their w+-eye color and were crossed to a y w strain. Wild type activity of the Hmr-HA and Hmr-FLAG transgenes was tested for complementation of an Hmr rescue mutation in hybrids as done previously for +Hmr transgenes −Df(1)Hmr, y w v/FM6; ø{mel-Hmr-HA}/+ females to 501D. simulans w males. We recovered 193 501/Y; +/+w hybrid males but only 1 501/Y; ø{mel-Hmr-HA}/+w hybrid male, demonstrating that the transgene is +Hmr. Likewise, we crossed −Df(1)Hmr, y w v; ø{mel-Hmr-FLAG}/+ females to D. simulans v males, and recovered 451 v females, 258 w males and only 3 +w males.φC31-mediated transgenesis was performed by Genetic Services using the KOLhr was outcrossed to 1118w for six generations. Sibling crosses were then used to generate a homozygous 1118w; KO/LhrKOLhr (abbreviated as KOLhr), a heterozygous KO/+Lhr, and a wildtype 1118w; +/Lhr+Lhr line (abbreviated as +Lhr). All experiments with Lhr in this paper use these matched mutant and sibling controls unless otherwise specified. The 1D. simulans Lhr allele is caused by an insertion in the 5′ UTR and appears to make no transcript by RT-PCR 1Lhr was outcrossed to the inbred wild-type line 501w for 3 generations to generate the stock 501; Lhr1w (abbreviated as 1Lhr) and 501w, +Lhr (abbreviated as +Lhr). Lhr-HA transgenes were described previously y w F10 was created by single-pair matings between siblings for 10 generations.3P{EPgy2}Hmr allele that is marked with +y and +w described in 3Hmr. −Df(1)Hmr, y w v, abbreviated as −Df(1)Hmr, is described in Hmr RNA-Seq experiments, the 3Hmr stock and the transgenic lines (mel-Hmr-FLAG and sim-Hmr-FLAG) were outcrossed to y w F10 for 6 generations and then made homozygous.We refer to the KOLhr and KO/+Lhr sibling females, obtained from crosses of KO/+Lhr at 27°C, were crossed to two 1118w males. Flies were transferred to a fresh vial every 5 days for 15 days. Vials in which either the female or both males were missing or dead were not scored or transferred. To create the heteroallelic siblings KO/Df(2R)BSC44Lhr and KO/SM6aLhr, KO/LhrKOLhr were crossed to the −Lhr deletion stock Df(2R)BSC44/SM6aIndividual 1–2 day old virgin KO/+Lhr or KO/LhrKOLhr females were crossed to 1118w males at 27°C. Egg lays were carried out on grape juice/agar plates for 3 hour periods at either 2–3 days, 5–6 days or 10–11 days after eclosion of the female parents. The plates were maintained at 27°C and monitored over the next 24–36 hours for hatched eggs.3; +/+y w Hmr females were crossed to y w; ø{mel-Hmr-FLAG}/ø{mel-Hmr-FLAG} males. F1 males were crossed to −/FM6Df(1)Hmr; +/+ females to generate both 3/Df(1)Hmr−y w Hmr; ø{mel-Hmr-FLAG}/+ and 3/Df(1)Hmr−y w Hmr; +/+. Similarly, 3; +/+y w Hmr females were crossed to y w; ø{sim-Hmr-FLAG}/ø{sim-Hmr-FLAG} males. F1 males were crossed to −/FM6Df(1)Hmr; +/+ females to generate 3/Df(1)Hmr−y w Hmr; ø{sim-Hmr-FLAG}/+. Lastly, y w; +/+ females were crossed to y w; ø{mel-Hmr-FLAG}/ø{mel-Hmr-FLAG} males. F1 males were crossed to −/FM6Df(1)Hmr; +/+ females to generate the heterozygous wildtype control, y w/−Df(1)Hmr; +/+. These crosses were done at 27°C and in triplicate to generate 3 biological replicates.−Df(1)Hmr, y w v/FM7i, P{w+mC = ActGFP}JMR stock (abbreviated as −/FM7iDf(1)Hmr, GFP) was described previously +Hmr genotype, y w v/FM7i, P{w+mC = ActGFP}JMR  was created by crossing y w v/Y males with −/FM7iDf(1)Hmr, GFP females. FM7i, GFP/Y males from this +Hmr stock were then crossed to −/FM7iDf(1)Hmr, GFP females for 10 generations in order to make the autosomal backgrounds comparable between the two stocks.The −/FM7iDf(1)Hmr, GFP or +/FM7iHmr, GFP were crossed to v/Y D. simulans males. For each cross, 6 replicates were made each containing 25 0–12 hour-old virgin females and 50 4–6 day-old virgin males. Hybrid larval sons not carrying the balancer were selected by their −y mouth hook and −GFP body phenotypes. Additionally, some crosses were allowed to develop to ensure that only −Df(1)Hmr crosses produced hybrid sons. To generate D. melanogaster samples, 3 replicates of 10 −/FM7iDf(1)Hmr, GFP or +/FM7iHmr, GFP virgin females were crossed to 15 FM7i, GFP/Y males. Larval sons not carrying the balancer were selected by −y and −GFP phenotypes. To generate D. simulans samples, 3 replicates of 10 y w D. simulans virgin females were crossed to 15 v/Y D. simulans males. Larval sons were selected by −y.To generate hybrids, 2; 0.5% TritonX-100; 0.5 mM DDT) and then centrifuged for 5 minutes at 4°C. The pellet was washed with 500 µl buffer A1 and centrifuged. This process was repeated another two times. The pellet was lysed by douncing in 200 µl SDS lysis buffer . The lysate was allowed to rotate at 4°C for 20 minutes and then centrifuged. The supernatant was removed, quantitated using the Bradford assay and was run on an SDS-PAGE gel.50 mg of 1–17 hr embryo collections were dounced 30 times with a tight pestle in 500 ul buffer A1 , dialyzed down to 2M urea and injected into rabbits . The antisera was then purified by coupling purified His-Lhr to CnBr-activated Sepharose beads in the presence of 1% Triton-X and removing urea by dialysis. Antisera was eluted in 0.2 M glycine, pH 2.8 and then neutralized with 1M Tris, pH 8.5. The antibody failed to detect Lhr in immunofluorescent experiments but was used for Western blots in An Lhr cDNA was cloned into pDEST17 (Invitrogen). The expressed protein from 2, 2 mM Mg acetate2, 0.1% Triton X-100, 0.5 mM DTT, 0.5 mM PMSF) and homogenized with a dounce homogenizer. The homogenized lysate was centrifuged at 700 g for 10 minutes at 4° to pellet the nuclei. The supernatant was removed, the pelleted washed once in Buffer A, the nuclei centrifuged again and then resuspended in 1× embryo volume of Buffer MN . The nuclear lysate was sonicated briefly, micrococcal nuclease added to a concentration of 500 units/ml, and the chromatin digested for 1 hour at 4° with gentle agitation. EDTA and Triton X-100 were then added to a concentration of 5 mM and 0.1% respectively, to inactivate nuclease activity and solubilize the proteins, followed by incubation at 4° for 1 hour. After a second brief sonication, the digest was centrifuged at 12,000 g for 10 min at 4° and the supernatant was collected. 50 µl of the chromatin digest was diluted in IP Wash Buffer  with 1× protease inhibitor cocktail to a final volume of 125 µl per co-immunoprecipitation mixture. 15 µl of protein G-conjugated magnetic beads and 2–5 µl of antibody were added followed by incubation for 4 hours at 4° with gentle agitation. The beads were washed 3 times in IP Wash Buffer. The immunoprecipitated proteins were then eluted by boiling the beads in 1× Laemmli sample buffer for 5 minutes and analyzed by immunoblotting.0∼16 hour-old embryos were collected, dechorionated and snap frozen in liquid nitrogen. Embryos were then resuspended to 10× embryo volume of Buffer A  transcript levels were measured with primers from reference RNA extraction, cDNA synthesis and qRT-PCR assays were performed as in reference KOLhr and +Lhr female carcasses that were free of ovarian tissue. Primers Het-s2 and Het-as2 amplify from the coding sequence of HeT-AHeT-A copy number was normalized to RpL32  copy number using primers from reference The Invitrogen DNEasy kit was used to make genomic DNA from KOLhr and +Lhr was duplexed and run in a single lane. 51,193,832 filtered reads were obtained for +Lhr and 41,688,028 reads for KOLhr. Three biological replicates each of 501D. simulans w and 1Lhr ovarian mRNA libraries were run on a single lane and the number of filtered reads ranged from 36,472,726 to 43,449,879. For experiments with Hmr, two biological replicates were included for each genotype and all 8 samples were multiplexed in a single lane. The number of filtered reads for each sample ranged from 23,863,381 to 27,490,644. For larval samples, around 30 larvae were collected for each genotype and flash frozen in liquid N2. RNA was extracted from 2 biological replicates of each genotype using Trizol. Larval RNAseq libraries were generated and bar-coded using the TruSeq kit, and run in one lane of an Illumina HiSeq 2000 100 bp yielding 13,707,247 to 20,373,267 filtered reads per sample, except for one library which produced only 7,840,004 reads.For samples from ovaries, flies were kept at 27°C for several generations prior to and during the experiment. Freshly eclosed females were collected and aged 2–3 days and then transferred to fresh food with yeast paste for another 2–3 days. RNA was extracted, from ovaries dissected in chilled 1× PBS, using Trizol. Ovarian mRNA-Seq libraries were constructed at the Epigenomics Core Facility at Weill Cornell Medical College using the poly(A) enrichment method. Libraries were sequenced using the Illumina HiSeq2000 platform to produce 50 bp single reads which were then trimmed for quality and filtered to remove rRNA reads. One biological sample each from D. melanogaster genome using Tophat D. melanogaster genome from ENSEMBL. To find differentially expressed genes in D. simulans, we aligned reads to the D. melanogaster genome with Tophat, allowing two mis-matches. While this approach could potentially reduce mapping ability for diverged genes, it allowed us to take advantage of the better assembly and annotation of the D. melanogaster genome.Reads mapping to either rRNA or repetitive DNA were filtered out using Bowtie D. melanogaster and D. simulans genomes TAHRE, which has only a few insertions in the genome and is known to be absent from the reference genome since only two telomeres are included in the assembly D. melanogaster or 3 mismatches when aligning reads from D. simulans or hybrids, to a database consisting of the consensus sequences of the annotated TEs and repeats found in Repbase as well as de novo predicted TEs generated by piler-DF using the 12 Drosophila genomes To maximize the TEs considered in our analyses, we mapped reads to two different databases using Bowtie. First, reads were uniquely mapped to a database consisting of all the annotated TE insertions in the http://www.fruitfly.org/sequence/sequence_db/na_re.dros) which itself was constructed from GenBank sequences. This file includes some mis-annotated TEs and non-satellite sequences. We counted reads that mapped to these repeats without any mismatches and calculated statistical significance of differential expression among satellites with F.E.T. in the DEG-seq package.To analyze reads mapping to satellite DNAs, we built a database using a curated file from the Berkeley Drosophila Genome Project (KOLhr and +Lhr ovaries using the mirVANA kit (Invitrogen). Total RNA was size fractionated on a 15% Urea-PAGE gel to enrich for 18–29 nt small RNA, excised and eluted and then subjected to 2S rRNA depletion. This small RNA was ligated to a 3′ RNA adapter, gel purified, and then ligated to a 5′ DNA adapter. The adapter-ligated small RNAs were reverse transcribed and PCR amplified. The amplified PCR products were gel purified, quantified and sequenced in two lanes of a HISeq 2000 machine.Libraries were prepared as described but no oxidation was carried out KOLhr, and 120,143,855 piRNA reads and 36,388,192 siRNA reads in +Lhr.Only reads with a 3′ adapter were kept, which was then removed using a custom script D. melanogaster sequences from Repbase using Bowtie, allowing for one mismatch. Ping-Pong scores were calculated using reads mapped with up to 1 mismatch, as described in reference D. melanogaster sequences from Repbase with Bowtie, without allowing for any mismatches.piRNA reads were mapped uniquely to all Immunofluorescence and FISH were performed on embryos and ovaries as described in references Rat anti-HA antibody  was used at 1∶100, rat anti-Vasa (DSHB) was used at 1∶25, Fibrillarin  was used at 1∶100, anti-HP1a antibody  was used at 1∶100. Alexa fluorophore-conjugated secondary antibodies were used to detect the primary antibody. Fluorescently labeled probes against GA-rich satellites, AACAC, 2L3L, 359 bp and dodeca were obtained from Sigma with sequences described in references D. melanogaster Hmr was made by correcting 3 frame-shift errors in the RE54143 cDNA XbaI-HindIII fragment from the LD22117 cDNA, followed by replacement of a 2172 bp NdeI-ZraI fragment from the p83 genomic clone D. simulans Hmr CDS was PCRd out of cDNA and cloned into pENTR/D-TOPO. The Lhr plasmids and yeast two-hybrid destination vectors and assays are described in reference A full-length coding-sequence plasmid of Illumina sequence data from this study are available from the NCBI website under BioProject number PRJNA236022.Figure S1KO/+Lhr; LhrHA/+ ovaries. Arrows point to overlaps between bright FISH and HA-staining foci. (B) mel-Hmr-HA (green) colocalizes with GAGAA (red) and (C) dodeca (red) in nurse cells of 3Hmr; mel-Hmr-HA/mel-Hmr-HA ovaries in a subset of nuclei. Arrows point to overlaps between FISH signals and the brightly staining foci of mel-Hmr-HA. Two different egg chambers are shown for both dodeca and GAGAA.Lhr and Hmr colocalize with specific satellite sequences in ovaries. Nurse cell nuclei (blue) are stained with DAPI in all panels. Scale bars represent 5 µm. (A) mel-Lhr-HA (green) colocalizes with GAGAA and AACAC  in the nurse cells of (TIF)Click here for additional data file.Figure S2Hmr-FLAG transgenes. Hmr transcript levels in transgenic lines were compared to the host strain (−Hmr) and also to +/Hmr−. The transgenes are heterozygous, therefore both the transgenic lines and +/−Hmr carry one copy of +Hmr. RNA was isolated from ovaries and Hmr expression levels were normalized relative to RpL32. Error bars represent standard error within 3 biological replicates. The difference in the expression level of mel-Hmr-FLAG and sim-Hmr-FLAG is significant . Additionally, the expression of mel-Hmr-FLAG is significantly different than an endogenous copy of Hmr .qRT-PCR analysis of (TIF)Click here for additional data file.Figure S3KOD. melanogaster Lhr allele generated by homologous recombination. (A) Lhr and flanking genes are shown, the red triangle labeled +w indicates the site of the insertion in the KOLhr allele, which is predicted to be ∼4.7 kb based on the structure of the targeting vector. Products used in RT-PCR reactions in (B) are shown below the genes. EDTP gene is partial; w+ insertion not to scale. (B) RT-PCR from adult females shows no Lhr transcript spanning the +w insertion (Lhr-5′-w-3′) in KOLhr. A highly reduced amount of Lhr transcript is detected 3′ to the w+ insertion (Lhr-w-3′). The flanking genes Bap55 and EDTP are not affected. 1118w was used as a +Lhr control. +, − indicates presence or absence of reverse transcriptase (RT). (C) Western analysis shows that KOLhr produces no protein. A non-specific band indicated by the asterisk is used a loading control.The (TIF)Click here for additional data file.Figure S4HeT-A levels in Lhr mutants. qPCR was used to estimate the transcript levels of HeT-A relative to the gene RpL32 in poly-A primed cDNA samples obtained from ovarian RNA from two different −Lhr backgrounds and matching controls. (A) Ratio of HeT-A/RpL32 in KOLhr vs. +Lhr, showing mean from 3 biological replicates. Significance of fold change was calculated using Welch's one-tailed t-test; p<0.05. (B) Ratio of HeT-A/RpL32 in KO/Df(2R)BSC44Lhr vs. +/Df(2R)BSC44Lhr, showing mean from 4 biological replicates. Significance of fold change was calculated using the one tailed Wilcoxon rank sum test; p<0.05.qRT-PCR analysis shows elevated (TIF)Click here for additional data file.Figure S53Hmr; Hmr-HA (A–C) and mel-Lhr-HA (green) in KO/+Lhr; Lhr-HA/+ (D) colocalize with HP1A (red) at the telomere cap on polytene chromosomes. mel-Hmr-HA shows a range of distributions at the telomere, including punctate (B) and continuous across the chromosome terminus (C). Scale bar is 1 µm.Localization of Hmr-HA and Lhr-HA to the telomeres is independent of dosage of endogenous copies. mel-Hmr-HA (green) in (TIF)Click here for additional data file.Table S1KOLhr mothers have a reduced hatch rate. Hatching of eggs laid by KO/+Lhr or homozygous KOLhr mothers crossed to wild-type fathers was followed for 36 hrs after egg lay. For KO/+Lhr, 34 eggs from days 2–3, 289 from days 5–6 and 668 eggs from days 10–11 were counted. For KOLhr, 46 eggs from days 2–3, 209 from days 5–6 and 287 eggs from days 10–11 were counted. The significance of the difference in the hatch rates of the eggs laid by KOLhr and KO/+Lhr mothers was calculated by one tailed F.E.T., and was significant at all time points (p<10−4).Eggs laid by (DOCX)Click here for additional data file.Table S2KOLhr and +Lhr ovaries based on reads uniquely mapped with no mismatches to either the individual-insertion or consensus-sequence TE databases.DEG-seq output measuring the statistical significance of the differences in TE expression between (XLSX)Click here for additional data file.Table S3−Hmr and −/Hmr+Hmr ovaries based on reads uniquely mapped with no mismatches to either the individual-insertion or consensus-sequence TE databases.DEG-seq output measuring the statistical significance of the differences in TE expression between (XLSX)Click here for additional data file.Table S4+Hmr and −Hmr male larvae based on reads uniquely mapped with up to three mismatches to either the individual-insertion or consensus-sequence TE databases.DEG-seq output measuring the statistical significance of the differences in TE expression between (XLSX)Click here for additional data file.Table S5KOLhr and +Lhr ovaries, based on reads uniquely mapped with up to 2 mismatches.Cuffdiff output measuring the statistical significance of the differences in protein-coding gene expression between (XLSX)Click here for additional data file.Table S6−Hmr and −/Hmr+Hmr ovaries, based on reads uniquely mapped with up to 2 mismatches.Cuffdiff output measuring the statistical significance of the differences in protein-coding gene expression between (XLSX)Click here for additional data file.Table S7KOLhr and +Lhr ovaries, based on reads uniquely mapped with no mismatches.DEG-seq output measuring the statistical significance of the differences in satellite DNA expression between (XLSX)Click here for additional data file.Table S8−Hmr and −/Hmr+Hmr ovaries, based on reads uniquely mapped with no mismatches.DEG-seq output measuring the statistical significance of the differences in satellite DNA expression between (XLSX)Click here for additional data file.Table S9KOLhr and +Lhr ovaries.DEG-seq output measuring the statistical significance of the differences in piRNAs uniquely mapping with up to one mismatch to TE consensus sequences from Repbase between (XLSX)Click here for additional data file.Table S10KOLhr and +Lhr ovaries calculated as described in Ping-pong scores in (XLSX)Click here for additional data file.Table S11KOLhr and +Lhr ovaries.DEG-seq output measuring the statistical significance of the differences in piRNAs uniquely mapping with up to one mismatch to piRNA clusters between (XLSX)Click here for additional data file.Table S12KOLhr and +Lhr ovaries.DEG-seq output measuring the statistical significance of the differences in siRNAs uniquely mapping with no mismatches to TE consensus from Repbase between (XLSX)Click here for additional data file.Table S13501D. simulans w and 1Lhr ovaries, based on reads uniquely mapped to the D. melanogaster genome with up to 2 mismatches.Cuffdiff output measuring the statistical significance of the differences in protein-coding gene expression between (XLSX)Click here for additional data file.Table S14501D. simulans w and 1Lhr ovaries based on reads uniquely mapped to either the individual-insertion or consensus-sequence TE databases, while allowing for no mismatches for insertions and up to three mismatches for consensus.DEG-seq output measuring the statistical significance of the differences in TE expression between (XLSX)Click here for additional data file.Table S15D. melanogaster and D. melanogaster- D. simulans hybrids (Sheets A and B), and between D. simulans and D. melanogaster-D. simulans hybrids (Sheets C and D). Unique reads were mapped to the individual-insertion (Sheets A and C) and consensus-sequence (Sheets B and D) TE databases, allowing for up to 3 mismatches.DEG-seq output measuring the statistical significance of the differences in TE expression of male larvae between (XLSX)Click here for additional data file.Table S16−/YHmr hybrids and lethal +/YHmr dying hybrids. Unique reads were mapped to the individual-insertion and consensus-sequence TE databases, allowing for up to 3 mismatches.DEG-seq output measuring the statistical significance of the differences in TE expression between viable (XLSX)Click here for additional data file.


Hepatitis B virus is hyperendemic in Sudan. Our aim was to molecularly characterize hepatitis B virus from Sudanese individuals, with and without liver disease, because genotypes play an important role in clinical manifestation and treatment management.Ninety-nine patients - 30 asymptomatic, 42 cirrhotic, 15 with hepatocellular carcinoma, 7 with acute hepatitis and 5 with chronic hepatitis- were enrolled. Sequencing of surface and basic core promoter/precore regions and complete genome were performed.The mean ± standard deviation, age was 45.7±14.8 years and the male to female ratio 77:22. The median (interquartile range) of hepatitis B virus DNA and alanine aminotransferase levels were 2.8 (2.2-4.2) log IU/ml and 30 (19–49) IU/L, respectively. Using three genotyping methods, 81/99 (82%) could be genotyped. Forty eight percent of the 99 patients were infected with genotype D and 24% with genotype E, 2% with putative D/E recombinants and 7% with genotype A. Patients infected with genotype E had higher frequency of hepatitis B e antigen-positivity and higher viral loads compared to patients infected with genotype D. Basic core promoter/precore region mutations, including the G1896A in 37% of HBeAg-negative individuals, could account for hepatitis B e antigen-negativity. Pre-S deletion mutants were found in genotypes D and E. Three isolates had the vaccine escape mutant sM133T.Sudanese hepatitis B virus carriers were mainly infected with genotypes D or E, with patients infected with genotype E having higher HBeAg-positivity and higher viral loads. This is the first study to molecularly characterize hepatitis B virus from liver disease patients in Sudan. Hepadnaviridae is responsible for chronic infection of more than 240 million people worldwide  and [710(−) (711–691 from EcoRI site)] for the second round   years. All sera were HBsAg- and anti-HBc-positive; 12 were HBeAg-positive/anti-HBe-negative, 75 were HBeAg-negative/anti-HBe-positive, and 12 had neither HBeAg nor anti-HBe. All patients were HBV DNA-positive, with a median (interquartile range) (IQR) viral load of [2.8 (2.2–4.2)] log IU/ml and ALT median (IQR) level was [30 (19–49)] IU/L (reference range 5–40 IU/L) .The 99 cases were classified into five clinical groups: HCC n=15), CR ( n=42), ASCs (n=30), AH ( n=7) and CH ( n=5). The HCC patients were significantly older than patients in the other groups (p <0.05) and the AH patients were significantly younger (p <0.05). The ALT levels were the highest in the AH (p <0.05) , followed by phylogenetic analysis , followed by phylogenetic analysis (trees not shown) and thirdly,11 of 29 by RFLP analysis.Firstly, 53 of 99 were genotyped using amplification and sequencing of the complete S  could not be genotyped using any of the three methods. No sequences could be obtained for 10 of these isolates and for the remaining 8 only the BCP/PC region was sequenced, which is not sufficient to differentiate between genotypes D and E.The genotype distribution in the 81 samples was 59% genotype D: 30% genotype E: 8.5% genotype A: 2.5% putative D/E recombinant. If we include the 18 samples that could not be genotyped, 48.5% (48/99) were infected with genotype D, 24.2% (24/99) with genotype E, 7.1% (7/99) with genotype A and 2% (2/99) were infected with a putative D/E recombinant.Phylogenetic analysis of the complete S region allowed for further classification into subgenotypes Figure . Based odata not shown).The complete genome of four isolates was amplified, two (SDAC024 and SDAC031) belonged to genotype D and two (SDAC047 and SDAC125) to genotype E. Although the genotypes determined using the complete genome agreed with that determined using the S ORF, there was a discrepancy in the subgenotype classification of SDAC031. Using phylogenetic analysis of the complete S, it clustered as an outlier of subgenotype D4, whereas following complete genome phylogenetic analysis it clustered with D6  and HBV DNA levels in patients infected with genotype E were significantly higher (p <0.05) Table .Of the 70 isolates amplified and sequenced in the BCP/PC region, 62 amplfied in the S region and thus had genotype assignments. 57 isolates belonged to either genotype D or E, and 5 to genotype A. Seven of 8 genotype D or E isolates from HBeAg-positive patients had wild-type BCP/PC, and one (SDAC118) had G1764T. The mutations found in genotype D or E isolates from HBeAg-negative individuals are shown in Figure Although generally the BCP/PC region cannot be used to determine specific genotypes, genotype A and its subgenotypes can be differentiated from genotypes D or E because of specific sequence characteristics. One genotype A isolate from a HBeAg-positive patient, (SDAC108) had A1762T/G1764A. From the BCP/PC sequences it was deduced that this isolate belonged to subgenotype A2, because it had GCAC at 1809–1812, 1858C and 1888G. The other four genotype A isolates were from HBeAg-negative individuals. These isolates, SDAC062, SDAC073 SDAC100 and SDAC113, belonged to subgenotype A1 because they had TCAT at 1809–1812, 1858C and 1888A. One isolate had wild-type BCP/PC region, two had A1762T/G1764A, one with G1862T and the fourth isolate had G1862T alone. No significant association was found between the BCP/PC mutations and genotypes or clinical groups.EcoRI site), were found in two isolates (SDAC095 genotype E and SDAC098 genotype D). Three cis-acting elements mutations were found: C1092T in the Enhancer I in one isolate, C1155T in the Enhancer I in 6 isolates and T3109G S2 promoter in 2 isolates. PreS2F22L was found in 6 genotype E and three genotype D isolates. The vaccine escape mutation, sM133T, was observed in genotype E isolates, SDAC022, SDAC067 and SDAC088. In the reverse transcriptase domain of the polymerase, the following mutations were detected: rtA194T in SDAC049 (genotype D), rtV207M in SDAC020 (genotype D), rtS213T in SDAC063 and SDAC098 (genotype D), SDAC002, SDAC121 and SDAC004 (genotype E), rtV214A in SDAC059 (genotype E) and rtS215Q in SDAC049 and SDAC090 (genotype D).The complete S region was sequenced for 53 isolates and pre-S mutations were detected in 14 (26%). Four mutational patterns were identified: pre-S1 deletions , pre-S2 start codon mutation , pre-S2 start codon mutation with pre-S2 deletion  and pre-S2 deletion alone   . In agreIn agreement with the study in Sudanese blood donors , the preDiscrepant results were obtained in the subgenotype assignment of SDAC031 when the complete S and complete genome were analyzed. The complete S clustered as an outlier of D4 whereas the complete genome clustered with D6. D4 and D6 are phylogenetically closely related Figure . Furtherin utero[The HBV genotypes show a distinct geographical distribution in Africa, with genotype D predominating in the North, genotype E in the West and genotype A in the South-East . This isin utero. Thus bain utero. The higin utero. The higin utero, whereasThe majority of the HBeAg-negativity was as a result of the classical G1896A, which abolishes HBeAg expression  and occuFour different pre-S mutational patterns were identified Table . In genoin vitro resistance test system, the mutants showed no resistance to lamivudine (LMV), entecavir (ETV), adenofovir (ADF) and tenofovir (TDF). .Six genotype E and three genotype D isolates had preS2F22L, which is a risk factor for HCC ,51. InteThis is the first study to molecularly characterize HBV from Sudanese liver disease patients, who were predominantly infected with genotypes D and E, allowing comparison of the effect of these genotypes on clinical manifestation in the same ethnic group.AH: Acute hepatitis; ALT: Alanine amino transferase; ASC: Asymptomatic carriers; BCP/PC: Basic core promoter/precore; CH: Chronic hepatitis; CRF: clinical report form; DFG: Deutsche Forschungsgemeinschaft (German Research Foundation); DNA: Deoxyribonuclease; HBcAb: Hepatitis B core antibody; HBeAb: Hepatitis B e antibody; HBeAg: Hepatitis B e antigen; HBsAb: hepatitis B surface antibody; HBsAg: Hepatitis B surface antigen; HBV: hepatitis B virus; HCC: Hepatocellular carcinoma; IQR: Interquartile range; CR: cirrhosis; ORF: open reading frame; PCR: Polymerase chain reaction; RFLP: Restriction fragment polymorphism; rt: Reverse transcriptase; s: Surface; sp: Spacer; WHO: World Health Organization.The authors declare that there are no competing interests.Conceived the study: AK, DG, HM; Designed the experiments: MY, DG, AK; Performed the experiments:MY; Analyzed the data: MY, AK; Contributed reagents/materials: AK, SB, HM; Wrote the paper: MY, AK Read, contributed to and approved the paper: MY, HM, SB, DG, AK. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/328/prepub


Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4′-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicidalpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-α were released. This study provides a feasible way to produce interesting lipid A species in E. coli.The lipid A moiety of  O-antigen repeats. Lipid A, the hydrophobic anchor of LPS, is responsible for the bioactivity of LPS [Lipopolysaccharide (LPS), known as endotoxin, is the prominent constituent in the outer leaflet of outer membranes in most Gram-negative bacteria ,2,3 and y of LPS . It can y of LPS ,7 and gey of LPS .Escherichia coli lipid A  were constructed by integrating and/or deleting key genes related to lipid A biosynthesis and modification in the chromosome. The structures of lipid A in these mutants were confirmed by thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI/MS). All mutants grew as well as the wild-type, and their LPS induces less TNF-α than the wild-type E. coli LPS when used to stimulate murine macrophage RAW264.7 cells. This study provides a convenient method to produce novel lipid A vaccine adjuvant in E. coli.In this study, E. coli . Lipid A isolated from HW000 showed a similar pattern as that from W3110. In the spectrum of lipid A isolated from HW001, there is only one major peak at m/z 1716.3, suggesting that the lipid A in HW001 is completely dephosphorylated at the 1-position. In the spectrum of lipid A isolated from HW002, there is only one major peak at m/z 1506.1, suggesting that lipid A isolated from HW002 is P-MPLA. Both TLC and ESI/MS data indicate that HW001 can convert all lipid A into MPLA and HW002 can convert all lipid A into P-MPLA. This indicates that the gene lpxE inserted in the chromosome of HW001 and HW002 was well expressed and LpxE can modify the structure of lipid A in vivo and that lpxM encodes the only late acyltransferase in E. coli that adds a secondary acyl chain to the 3′-position of lipid A.Lipid A isolated from W3110, HW000, HW001 and HW002 were further analyzed by ESI/MS . The lipE. coli O111:B4 LPS was used as a positive control, and PBS  was used as a negative control.LPS was purified from W3110, HW001 and HW002 and used to stimulate macrophage cell line RAW264.7. Levels of TNF-α released by the cells after stimulation were examined . E. coliThe levels of TNF-α released by RAW264.7 cells were proportional to the concentration of LPS up to 100 ng/mL, but there was little difference between LPS concentrations of 100 ng/mL and 1000 ng/mL. At all concentrations, lower levels of TNF-α were released when RAW264.7 cells were stimulated by HW001 LPS and HW002 LPS than by the controls O111:B4 LPS or W3110 LPS . The lowE. coli LPS, suggesting they are good candidates for vaccine adjuvant development. The changes in the number of the acyl chains and the phosphate groups of lipid A might decrease the binding ability by TLR4/MD-2 and lead to the lower release of TNF-α.These results demonstrated that LPS from HW001 and HW002 induced lower secretion of TNF-α than that of wild-type Restriction enzymes, shrimp alkaline phosphatase, T4 DNA ligase and DNA ladder were purchased from Sangon . Plasmid DNA was prepared by using the EZ-10 spin column plasmid mini-preps kit from Bio Basic Inc. . PCR reaction mixtures (50 μL) used in this study contain 5 μL 10× Ex Taq buffer, 4 μL dNTP mixture (2.5 mM each), 1 μL plasmid template (100 ng/μL), 1 μL forward primer (20 μM), 1 μL reverse primer (20 μM) and 0.5 μL TaKaRa Ex Taq DNA polymerase. The PCR reaction was first heated to 94 °C and maintained for 10 min, followed by 35 cycles of denaturation (94 °C for 30 s), annealing (30 s at 55 °C or 58 °C) and extension (72 °C for 3 min). At the end, an additional 10 min incubation at 72 °C was used. PCR products were purified by using the TIANgel midi purification kit from Tiangen . Primer synthesis and DNA sequencing were performed by Sangon. The sequences of primers used in this study are listed in All bacterial strains and plasmids used in this study are listed in lacIU-loxp-kan-loxp-lacID, in the vector pBlueScript II SK+. Primers lacIU-F and lacIU-R were used to amplify the upstream fragment of lacI; primers lacID-F and lacID-R were used to amplify the downstream fragment of lacI; primers kan-loxP-F and kan-loxP-R were used to amplify the fragment loxp-kan-loxp from pMG2-kanr [kan-loxpRE, in the vector pBlueScript II SK+. Fragments loxpLE and loxpRE were obtained by the annealing of the primers loxpLE-F, loxpLE-R and loxpRE-F, loxpRE-R, respectively, according to the method of Arakawa [kan was PCR amplified from pK18mobsacB [kan-F and kan-R. Plasmid pBS-Mkan was constructed by inserting a fragment, lpxMU-loxpLE-kan-loxpRE-lpxMD, in the vector pBlueScript II SK+. Primers lpxMU-F and lpxMU-R were used to amplify the upstream fragment of lpxM; primers lpxMD-F and lpxMD-R were used to amplify the downstream fragment of lpxM, and primers kan-loxpLE-F and kan-loxpRE-R were used to amplify the fragment loxpLE-kan-loxpRE from pDTW202. Plasmid pKD-Cre was constructed by replacing the λ Red genes with the gene cre in the vector pKD46 [cre was amplified from the vector pSH47 [cre-F and cre-R, digested with SacI and NcoI and ligated with the vector pKD46, which is similarly digested. The gene cre was put under the arabinose-inducible ParaB promoter. The plasmid pKD-Cre has a temperature sensitive replicon.Four plasmids pBS-Ikan, pDTW202, pBS-Mkan and pKD-Cre were constructed in this study a. Plasmi Arakawa , and the8mobsacB  with prior pKD46 . The genor pSH47 , using pE. coli mutants, HW000, HW001 and HW002, were constructed by deleting and/or integrating genes in the chromosome of E. coli  sites flanking the kan gene and excises the kan gene, resulting in the lacI mutant HW000. Secondly, the DNA fragment lacZU-lpxE-FRT-kan-FRT-lacZD was amplified from pWSK29-FnlpxE-Fkan [lacZ-F and lacZ-R and transformed into HW000/pKD46. Under the help of Red enzymes expressed by pKD46, the lacZ gene was broken by the inserted DNA fragment lpxE-FRT-kan-FRT. The kan gene was then eliminated by transforming pCP20. Plasmid pCP20 expresses the FLP recombinase, which acts on the directly repeated FRT (FLP recognition target) sites flanking the kan gene and excises the kan gene [lpxMU-loxpLE-kan-loxpRE-lpxMD was amplified from pBS-Mkan using primers lpxMU-F and lpxMD-R and transformed into HW001. Under the help of Red enzymes expressed by pKD46, the lpxM gene in the chromosome was replaced by the DNA fragment loxpLE-kan-loxpRE. The kan gene was then eliminated by transforming pKD-Cre, resulting in the triple mutant HW002. Since the short sequences loxp left by deleting lacI, loxpLR left by deleting lpxM and FRT left by inserting lpxE are different, they could not recombine with each other in the double or triple mutants.Three  E. coli b. FirstlpxE-Fkan  using prkan gene , resulti600 of 1.0. The cells were harvested by centrifugation at 8000 rpm for 20 min and washed with PBS. The final cell pellet was resuspended in a single phase Bligh-Dyer mixture [Typically, 200 mL cultures were inoculated from overnight cultures and grown to an OD mixture  (76 mL) The dried lipid A was dissolved in chloroform/methanol (4:1 v/v) and spotted onto a silica gel 60 TLC plate. The plate was developed in the solvent of chloroform/methanol/water/ammonia (40:25:4:2 v/v/v/v). After drying and spraying with 10% sulfuric acid in ethanol, the separation of lipid A on the plate could be visualized by charring at 175 °C.For ESI/MS analysis, dried lipid A was dissolved in chloroform/methanol (4:1 v/v) and subjected to ESI/MS in the negative ion mode. The mass spectra were acquired on a Waters SYNAPT Q-TOF mass spectrometer equipped with an ESI source. Data acquisition and analysis were performed using MassLynx V4.1 software .E. coli strains using the phenol-water procedure [E. coli cells (3 g) were suspended in water (10 mL) at 68 °C, and 90% aqueous phenol (10 mL) was added. The mixture was stirred for 1 h at 68 °C, then cooled to 4 °C and centrifuged at 4000 rpm for 20 min to separate the water and phenol phases. The water phase was collected, dialyzed against water and lyophilized. The crude LPS (0.5 g) was resuspended in 100 mM Tris-HCl  and then by treating with DNase I, RNase A at the concentration of 100 and 50 μg/mL at 37 °C for 2 h, respectively. Then 100 μg/mL proteinase K at 37 °C for 2 h was performed. For further purification, 5 mL water-saturated phenol was added to the crude LPS sample. The mixture was centrifuged at 4000 rpm for 20 min. The water phase was collected, dialyzed and lyophilized. Phospholipid contaminants in the sample were removed by extraction with chloroform/methanol (2:1 v/v) [LPS were isolated from rocedure . Wet E. 2:1 v/v) . Purifie2:1 v/v) .E. coli O111:B4 LPS was purchased from Sigma-Aldrich. The murine macrophage cell line RAW264.7 was obtained from the cell bank of the Chinese Academy of Science . It was maintained at 37 °C with 5% CO2 in DMEM  containing 10% FBS , supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were cultured at 105/well in 96-well plates with 200 μL medium each well. After 18 h, the RAW264.7 cells were stimulated with increasing concentration of LPS . 24 h later, culture supernatants were collected, centrifuged to remove the contaminates and stored at −80 °C for cytokine analysis. Concentrations TNF-α were determined using the enzyme-linked immunosorbent assay kit (eBioscience), according to the manufacturer’s instruction.p < 0.05 was considered a significant difference and p < 0.01 was considered extremely significant difference. All data were analyzed with SPSS Statistics 17.0.One-way analysis of variance was performed to determine the statistical significance of the differences between mean values for various experimental and control groups. Data were expressed as means ± standard deviation, and the experiments were performed in triplicate. The means were compared using the least significant difference test. Salmonella Minnesota R595 and then removing the 1-phosphate group chemically. This MPLA product was 0.1% as toxic as wild-type LPS without affecting the immune-stimulating activity when tested in pre-clinical rabbit pyrogenicity assays [S. minnesota makes multiple lipid A species, which are difficult to separate from each other [MPLA is safe and effective in eliciting Th1 response to heterologous proteins in animal and human vaccines ; therefoy assays ,32. Howech other .E. coli, the most common bacterium used for gene engineering, synthesizes only one type of lipid A. Therefore, we constructed two E. coli strains, HW001 and HW002, that could produce MPLA, by deleting or constitutively expressing genes related to lipid A biosynthesis in the chromosome. The gene lpxE encoding a phosphatase LpxE was integrated in the chromosome of HW001. LpxE can remove the phosphate at the 1-position of lipid A and has been used to develop Salmonella vaccines [lpxM in the chromosome of HW001. The lpxM gene product is an important virulence factor in a murine model of E. coli pathogenicity [Salmonella lpxM mutants produce pentaacylated lipid A with reduced affinity to TLR4, resulting in virulence attenuation [However, vaccines . HW002 wgenicity , and Salenuation . As expeE. coli using plasmids as the vector, but plasmids are not stable in bacteria and usually need antibiotics as selection markers. The markerless chromosomal integration of lpxE in HW001 and HW002 make these strains ideal for industrial production of MPLA. Other than LpxE and LpxM, there are enzymes, such as PagL and PagP, which could also modify the structure of lipid A to change its interaction with TLR4/MD-2 [Heterologous genes are usually expressed in LR4/MD-2 .


Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity.The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in  RNA viruses are ideal systems for the study of population dynamics, relationships among pathogen traits such as fitness and virulence, and of host immune responses to pathogen attacks. Based on experimental evolution studies, early models equated parasite virulence with fitness. Some reports showed that viral virulence and fitness can be unlinked. Here we present evidence that the highly disordered N-terminal region of a potyviral P1 protein negatively regulates its self-cleavage activity. Removal of this regulator domain greatly affects viral infection, which is characterized by accelerated early replication and enhanced symptom severity. These properties are nonetheless associated with low viral accumulation and high induction of antiviral resistance markers. Finally, we propose that host-dependent regulation of P1 processing efficiency modulates viral virulence and alleviates the host antiviral responses. Due to the limited coding capacity of their genome, viruses undertake distinct translational strategies Potyvirus (family Potyviridae) belong to the picorna-like supergroup and represent one of the largest groups of plant-infecting RNA viruses cis cleavage at their respective C-termini trans cleavage activity Members of the genus Plum pox virus (PPV) as a model system to study potyviral P1. Our results indicate that P1 and its protease domain have a positive role in potyvirus infection, independent of silencing suppression. Moreover, the in vitro and in planta data presented here suggest that host-dependent regulation of P1 self-cleavage activity has evolved to modulate the efficiency of viral infection and escape plant defense responses.Here we used Turnip mosaic potyvirus (TuMV) defective in RNA silencing suppression activity was reported in the Arabidopsis thaliana triple mutant line dcl2-1 dcl3-1 dcl4-2 (dcl2/3/4) Nicotiana clevelandii and able to infect herbaceous hosts Arabidopsis mutant plants with defective antiviral silencing pathways. At 15 days post-agro-inoculation (dpi), ΔP1-infected Col-0 and dcl2/3/4 plants were almost symptomless, whereas PPV-infected Col-0 plants showed leaf chlorosis, which was stronger in the dcl2/3/4 mutant line (Tobacco etch virus (TEV) dcl2/3/4 line did not restore the ΔP1 phenotype and viral accumulation to wild-type PPV levels  system. Replacement of catalytic S259 (S) and P2 and P1 cleavage site residues HY-307,308 (HY) with alanine impaired P1 protease self-cleavage, precluding release of the mature 35.3 kDa P1 protein and the 11.1 kDa HCPro fragment (HC-97) (Thosea asigna virus 2A (T2A) “self-cleaving” peptide To study the relevance of P1 motifs in protease activity, we mutated selected PPV P1 conserved amino acids to alanine . The vir (HC-97) . No prot (HC-97) , in accoin vitro translation were introduced into binary vectors bearing the full-length PPV cDNA. N. clevelandii plants were agro-infiltrated with Agrobacterium strains harboring pSN-PPV, pSN-PPV ΔP1, or pSN-PPV plasmids with point mutations in P1, and GFP fluorescence was monitored to follow the infection process of the derived viral clones. Infectious clones with full deletion (ΔP1) or point mutations in the P1 sequence (FG and WE) had characteristic ring-shaped GFP foci with no detectable fluorescence in the center compared to the wild-type virus (PPV) (Potato virus A (PVA) clone with the GFP gene inserted into the P1 region in vitro translation assays, did not show GFP fluorescence, infection symptoms or CP accumulation  . A similin vitro. A virus harboring the same mutation was infectious, however, and able to move systemically, with a marked delay compared to the parental virus Although P1 VE-189,190-AA replacement led to no detectable cleavage in WGE experiments , a faintAt the same time point at which P1 VE-189,190-AA reversion was detected (22 dpi), the rest of the mutations introduced were stably maintained in both FG and WE infectious clones , confirming that mutations of N-terminal motifs are less detrimental than protease defects.N. benthamiana. Leaves were co-infiltrated with an Agrobacterium strain bearing p35S:GFP as a silencing reporter and Agrobacterium strains containing the PPV silencing suppressor HCPro preceded by the wild-type P1 (pWT), by P1 with an alanine replacement of the catalytic S259 (pS) or by P1 S259A plus T2A (pST2A). GFP fluorescence was visible in all agroinfiltrated patches at 3 days post-agro-infiltration (dpa) (not shown). At 6 dpa, there were no differences between the empty control and the S construct. In contrast, bright fluorescence as a result of silencing suppression was maintained when HCPro was effectively released by P1 protease activity or by the ribosome skipping mechanism of T2A , using wild-type PPV as control. As predicted, wild-type PPV infected 16 of 16 challenged plants of both the mutant line and its wild-type background, at 23 dpi. Although we could not detect replication of the S mutant in Col-0 by GFP fluorescence or western blot analysis, GFP fluorescence was observed in 11 of 16 dcl2/3/4 plants  systems. The full-length P1 (WT) construct released the mature 35.3 kDa P1 protein in WGE but not in the RRL system . Viral amount at the end of the growth curve was further assessed by anti-PPV CP western blot, and confirmed that significantly less P1Pro virus accumulated than wild-type PPV at 148 hpi  or by the P1 lacking amino acids 2–163 (pP1Pro), as evaluated by GFP FI and western blot analysis . After removal of the signal peptide, this protein has a reported molecular weight of 34.8 kDa de novo (N. clevelandii). Based on MS/MS spectra, we verified that all five matched a glucan endo-1,3-β-D-glucosidase, EC 3.2.1.39, belonging to class II of the PR-2 family At the protein level, P1Pro-infected plant extracts were characterized by a specific band migrating at ≈35-37 kDa, which was unappreciable in wild-type PPV protein extracts and was thus analyzed by mass spectrometry (MS) . In the  de novo , since dTobacco mosaic virus (TMV) N. clevelandii plants  treatment induces class II PR-2 and class II PR-3 transcription N. benthamiana, while wild-type PPV-infected plants appeared almost symptomless, PPV P1Pro-infected plants showed extended chlorosis and necrotic lesions, similar to those observed in N. clevelandii  P1Pro-induced chlorosis was more accentuated than in wild-type PPV-infected plants , (ii) alBesides the self-cleavage activity intrinsic to its C-terminal end, other activities of potyviral P1 and its real contributions to viral replication remained vague, so much so that P1 earned the appellative “mysterious protein” in a recent review Arabidopsis dcl2/3/4 complementation data demonstrate a major function for P1 independent of RNA silencing suppression. The ring GFP phenotype of P1 mutants might thus be related not only to a silencing suppression defect, but also to other altered functions. Recently, the silencing suppressor-enhancing effect of P1 was attributed to cis elements that improve translation efficiency, rather than to complementary activity of the P1 protein In concomitance with the discovery that HCPro inhibits posttranscriptional gene silencing, it was suggested that P1 could act in conjunction with and strengthen the silencing suppressor activity of HCPro Our results nonetheless show that P1 is unquestionably involved in RNA silencing by limiting HCPro function. Deficiencies in polyprotein processing at the P1-HCPro junction abrogated PPV infectivity in wild-type plants, as also reported for TEV In silico analysis showed that the limited primary sequence conservation of the P1 N-terminal region is associated with residues predicted to be part of intrinsically disordered loops. The relevance of unfolded regions in the proteolytic maturation of viral polyproteins has been shown in vitro translation only in the WGE system, but not in RRL. In consequence, it was suggested that a non-viral factor, present in WGE and absent in RRL, might be involved in correct folding of the protease domain or presentation of the cleavage site Of the mature proteins encoded by the potyviral genome, P1 presents the greatest variability in length and in amino acid sequence In the context of viral infection, removal of the P1 protease antagonistic extension accelerated early viral replication and was followed by enhancement of symptom severity, with the appearance of plant stunting and necrotic lesions that did not characterize wild-type PPV. The greater aggressiveness did not parallel viral loads, however, as viral RNA and CP levels in plants systemically infected with the deletion mutant were lower than in wild-type PPV-infected plants. Lack of positive correlation between symptom severity and fitness is reported for several viral systems, including potyviral infections Considering that (i) defects in P1 self-cleavage preclude viral viability, (ii) viral RNA silencing suppression is impaired by the lack of separation between P1 and HCPro, (iii) P1 residues 1–164 are predicted to be mainly disordered, and negatively affect P1-HCPro processing in RRL, (iv) PPV early amplification dynamics are enhanced by removal of the P1 N-terminal end, (v) increased defense responses are associated with deletion of the P1 N-terminus, and (vi) removal of the P1 N-terminus reduces viral accumulation, we propose a model by which P1 acts to fine-tune potyviral replication by sensing specific host effectors. The presence of these cofactors leads to activation of the P1 protease domain and release of the silencing suppressor HCPro. The virus can thus effectively counteract host antiviral RNA silencing defenses and replicate successfully, but it can also stimulate additional defense responses. In an alternative scenario in which the cofactor is limited, P1-HCPro separation is restricted, RNA silencing suppression activity is disturbed, and some decrease in viral replication can therefore be predicted. We speculate that this mechanism holds viral replication below a level that would be detrimental to the host cell and reduces triggering of host immune responses, thus maintaining higher long-term replicative capacity.Bovine viral diarrhea virus (BVDV), which is dependent on the host chaperone Jiv An example of restriction in a virus infection step to avoid host defense responses is the regulation of autoprocessing of the NS2 protein of the pestivirus To understand the regulation of P1-HCPro processing, identification of the P1 activating host effector will be a priority for future studies. Moreover, P1 protease multifunctionality was postulated Potyviral P1 sequences were visualized and analyzed using Jalview Nicotiana clevelandii, N. benthamiana, N. benthamiana NahG Arabidopsis thaliana Col-0 and its triple mutant line dcl2-1 dcl3-1 dcl4-2Nicotiana plants were grown in a greenhouse maintained at a 16 h light/8 h dark photoperiod, temperature range 19–23°C. Arabidopsis plants were grown in the greenhouse or in vitro (see conditions below).in vitro translation and agro-infiltration constructs, can be found in A full-length cDNA copy of a PPV isolate In vitro transcription reactions were performed with the T7-Scribe Standard RNA IVT kit (CELLSCRIPT), including final DNase I digestion. RNA was purified by organic extraction/ammonium acetate precipitation. Quality was assessed by NanoDrop (Thermo Fisher Scientific) and by gel electrophoresis, with final concentration adjusted to 1 µg/µL. In vitro translation was carried out in the presence of L-[35S]methionine and L-[35S]cysteine (PerkinElmer) using the WGE or the RRL translation system (Promega), according to manufacturer's instructions. Samples were resolved in 12% tricine-SDS-PAGE Arabidopsis seeds were surface sterilized, kept at 4°C for three days, and sown in vitro on Murashige and Skoog medium Agrobacterium cultures were induced as described 600 was adjusted to 1.5, forceps were dipped into the culture, and the two youngest plantlet leaves were pierced. Forceps were flame-sterilized between cultures of different bacterial clones. Plates were sealed with gas-permeable tape (Millipore) and maintained in a growth chamber at 16 h light/8 h dark photoperiod, temperature 21±1°C, 60% relative humidity.N. benthamiana and N. clevelandii plants with A. tumefaciens strain C58C1 bearing the indicated plasmids, were as described N. clevelandii plants following reported agroinfiltration and sampling guidelines Agrobacterium was used.The transient RNA silencing assay and agro-infiltration of GFP fluorescence was monitored under long-wavelength UV light  and photographed using a Nikon D1X digital camera with a 62E 022 filter. Alternatively, images were acquired under a MZ FLIII epifluorescence microscope (Leica) using a GFP3 filter  and photographed with an Olympus DP70 digital camera. When indicated, GFP fluorescence was acquired by laser scanning .GFP fluorescent intensity quantification was carried out placing individual 5.0 mm-diameter leaf discs in a black 96-well plate. Signals were acquired in an Appliskan (Thermo Fisher Scientific) or Victor X2 (PerkinElmer) plate readers with the following settings: measurement time 500 ms, excitation and emission wavelengths of 485/10 nm and 535/20 nm, respectively.Plant tissue was ground in a mortar in liquid nitrogen or homogenized in a TissueLyzer bead mill (Qiagen). Total proteins were extracted in 150 mM Tris-HCl pH 7.5, 6 M urea, 2% (w/v) SDS, 5% (v/v) glycerol and 5% (v/v) β-mercaptoethanol; heat denatured  and centrifuged  to remove cell debris. Protein samples were separated by glycine-SDS-PAGE and electroblotted onto a nitrocellulose membrane. Ponceau red staining was used to control protein loading equivalence. Proteins were detected using anti-PPV CP and anti-PPV HCPro rabbit sera, and anti-GFP monoclonal antibody  as primary antibodies. Antibodies raised against tobacco class II PR-2 Total RNA was extracted with the FavorPrep Plant Total RNA Mini kit (Favorgen). Fragments spanning the PPV 5′UTR, and the coding sequences of P1 and the HCPro N-terminus were amplified with the Titan One Tube RT-PCR kit (Roche) using primers 1595_F/1597_R  and only peptides with an individual ion score above the identity threshold were considered correctly identified.Plant total protein extracts were separated by glycine-SDS-PAGE and stained with Coomassie blue. Gel bands of interest were excised manually, reduced, alkylated and in-gel digested. The tryptic-eluted peptides were subjected to MALDI-TOF/TOF analysis. Data were automatically acquired in an ABi 4800 MALDI TOF/TOF mass spectrometer (AB Sciex) and searched against NCBI non-redundant protein database, NCBInr_20121116. Mass tolerance for precursors was set to ±50 ppm and for MS/MS fragment ions to ±0.3 Da. The confidence interval for protein identification was set to ≥95%  Manual peptide N. benthamiana NahG plants were agro-inoculated with two independent bacteria cultures per each viral cDNA clone ; upper non-inoculated leaves were collected from 8 plants per culture (12 dpi). Total protein extracts were prepared as described for western blot assays and further purified by methanol/chloroform precipitation. Protein pellets were resuspended in 6 M guanidine hydrochloride and 100 mM HEPES, pH 7.5, and concentration was determined by RC DC assay (BioRad). Equal amounts of protein for each condition were trypsin-digested and labeled with iTRAQ Reagent Multi-plex kit (AB Sciex). Tags 114 and 116 were used for P1Pro biological replicates and 115 and 117 for wild-type PPV biological replicates. Labeled peptide samples were combined and subjected to LC-MS/MS analysis  using a nano liquid chromatography system (Eksigent Technologies) coupled to a Triple TOF 5600 mass spectrometer (AB Sciex). MS and MS/MS data were processed using Analyst TF 1.5.1 software (AB Sciex) and searched using the Mascot Server v. 2.3.02 (Matrix Science) against a customized database represented by N. benthamiana genome-predicted proteins (available at Sol Genomics Network p<0.05), and only peptides with an individual ion score above the identity threshold were maintained in quantification analysis. Proteins were considered differentially expressed if they had at least two quantified peptides and they were present in both P1Pro biological replicates with a false discovery rate <5%. LOG2 ratios relative to PPV biological replica #A (iTRAQ tag 115) were visualized by MultiExperiment Viewer N. benthamiana proteins were used as query in a WU-BLAST2 search against the TAIR10 protein dataset, to generate a list of homologous A. thaliana gene IDs. This was used as input in gene ontology term enrichment analysis using BinGO http://www.ncbi.nlm.nih.gov) accessions were used in the viral sequence analysis: YMV (YP_022752.1); PVV (NP_734369.1); PVY (NP_734243.1); VVY (YP_001931974.1); CaYSV (YP_003208051.1); JGMV (NP_734408.1); MDMV (NP_734143.1); SCMV (NP_734133.1); SrMV (CAC84438.1); PVA (NP_734359.1); TEV (NP_734207.1); ScaMV (NP_734123.1); TuMV (BAC02772.1). Plant GenBank accessions considered: N. tabacum class II PR-2 (P23547.1); N. tabacum class I PR-2 (AAA63541.1); S. tuberosum class II PR-2 (CAE52322.1); S. lycopersicum class II PR-2 (NP_001234798.1). N. benthamiana accessions can be found at Sol Genomics Network (ftp://ftp.solgenomics.net/genomes/Nicotiana_benthamiana/annotation/).The following GenBank Click here for additional data file.Figure S2Pseudo-reversions of P1 VE-189,190-AA mutations and infectivity of PPV ST2A viral clone.N. clevelandii plants were agro-inoculated with wild-type PPV and viral clone VE , clone S  and clone ST2A (P1 S259A+T2A peptide). (A) Anti-PPV HCPro (HC) western blot assay of upper non-inoculated leaves, collected at 22 dpi. For each clone, both samples shown in B) RT-PCR analysis of plants challenged with VE mutant clone (22 dpi). A PPV cDNA fragment encompassing the mutated region (nt 1–1197 of the PPV genome) was amplified by RT-PCR from a pool of two plants and analyzed by DNA sequencing. Sanger sequencing results of the RT-PCR fragment and of the inoculated plasmid DNA are shown. The wild-type PPV sequence is shown as reference. Nucleotides that differ from the wild-type sequence are indicated in lower case. The nucleotide that leads to reversion of the amino acid codon is underlined. Encoded amino acids are indicated beneath each nucleotide sequence and between box-drawing characters. Mutated residues are in italic , reverted residue underlined. (C) GFP-fluorescence pictures of upper non-inoculated N. clevelandii leaves were taken in an epifluorescence microscope (22 dpi). Scale bar, 1 cm. (D) Anti-PPV CP (CP) western blot assay of upper non-inoculated leaves (22 dpi); each lane corresponds to a single plant sample. Ponceau red-stained blot is shown as loading control.(TIF)Click here for additional data file.Figure S3HCPro silencing suppression activity is maintained in the absence of the P1 N-terminal region. Transient RNA silencing assay was done by co-infiltrating leaf tissue with an Agrobacterium p35S:GFP culture and cultures of Agrobacterium pSN.5 P1-S , pSN.5 PPV , or pSN.5 P1Pro . (A) Picture of N. benthamiana agro-infiltrated leaf was taken in an epifluorescence microscope (6 dpa). (B) GFP fluorescent intensity (FI) from N. benthamiana infiltrated leaf patches was quantified in a fluorometer and plotted using average pWT value at 6 dpa equal to 100. Line graph shows mean ± SD ; colors as in (A). For each time point, the mean GFP FI value (n = 16) from untreated leaves was used as blank. Analyzed time points (dpa) in the x axis. (C) Western blot detection of GFP protein in N. clevelandii samples (6 dpa); each lane represents a pool of samples from 2/3 infiltrated patches. N, non-infiltrated leaf sample. Ponceau red staining is shown as loading control. (D) Relative signal quantification for the immunoblot shown in (C) using average pWT value equal to 100. Histogram shows mean ± SD ; ns, not significant by Student's t-test.(TIF)Click here for additional data file.Figure S4Nicotiana clevelandii class II PR-2 peptide sequences assigned by MS/MS analysis. (A) Identified sequence of MALDI-TOF peptides labeled in de novo defined peptides, residues that differ from tobacco class II PR-2 isoform GI9 sequence (GenBank accession no. P23547.1) are in bold and underlined. Amino acids with near-/isobaric masses were bona fide assigned according to alignment consensus of the following panel. (B) Alignment of reference PR-2 sequences with N. clevelandii peptides . Residues that align with the peptides identified are boxed; background according the ClustalX color scheme N. benthamiana class II PR-2 predicted protein (Sol Genomics NbC25742659g0003.1); Stub_PR-2II, Solanum tuberosum class II PR-2 (GenBank CAE52322.1); Slyc_PR-2II, S. lycopersicum class II PR-2 (GenBank NP_001234798.1); Ntab_PR-2I, tobacco class I PR-2 clone gglb50 (GenBank AAA63541.1) as outlier. The last 22 residues of Ntab_PR-2I are hidden, as they do not align to the other accessions. In each alignment lane, positions of the first and last accession amino acids are indicated. In de novo defined peptides, residues that differ from the Ntab_PR-2II sequence are marked with a star.(TIF)Click here for additional data file.Text S1This file contains: Supplemental Methods; Supplemental References; Table S1, Virus species and GenBank accession numbers for viral protein sequences used in the study; Table S2, List of the primers used in the study.(PDF)Click here for additional data file.