Pseudotsuga menziesii var. menziesii (Mirb.) Franco) seed orchard was evaluated over four years. Using DNA microsatellite markers and bulk seed samples, we conducted pedigree reconstruction to assign each seed’s male and female parents, followed by determining the extent of pollen contamination , selfing rate, and, parental gametic contribution for each year. Overall, external pollen contamination rates ranged between 10 and 28%, selfing rate varied between 12 and 17%, and 80% of the seed crops were produced by 37–64% of the orchard’s parents. Pollination environment and seed crop size substantially influenced the observed results, particularly for small crops as pollen contamination was high in natural (28%) vs. intrusive pollination (10%). Generally, irrespective of the crop size, seed produced under natural pollination had higher pollen contamination, confirming the role of pollination environment manipulation in improving seed crops’ genetic quality.Seed orchards main function is delivering breeding programs’ gains in the form of genetically improved seedlings. They are unique experimental populations, perfectly suited for studying various pollination environments , affecting their mating system parameters. Here, under different pollination environment ), the mating system of a second generation, wind-pollinated, coastal Douglas-fir ( Seed orchards are of vital importance as they represent the vehicles connecting breeding and silviculture activities through packaging gain and diversity in their genetically improved seed crops. In order to fulfil this role, seed orchards are expected to function as closed, perfect panmictic populations2, an ideal scenario that is hardly met due to the commonly observed variation in reproductive success and phenology among parents as well as external gene flow (pollen contamination) from the ambient environment4. These factors could lead to reducing the genetic worth and diversity as well as harbouring higher levels of inbreeding in the resulting seed crops7. Pollen contamination has a drastic negative impact on both growth and adaptation reaching an estimated annual economic losses exceeding 9.4 million Canadian dollars for British Columbia’s advanced generations Douglas-fir seed orchards8.Tree selective breeding programs are designed to increase the economic values of future forests through planting superior stock with the highest genetic gain and diversity9 and supplemental-mass-pollination (SMP)10 were devised and implemented to enhance seed crops genetic quality. Bloom-delay, originally devised as fruit species frost-protection system11, has also been effective in slowing within seed orchards’ heat-sum accumulation relative to the ambient environment, thus delaying their reproductive development and acts as temporal isolation from the extraneous pollen12. An added feature of the bloom-delay treatment is compacting the reproductive phenology among seed orchard’s parents as heat-sum accumulation after treatment cessation is accelerated and hence speeds reproductive development leading to panmixia improvement13. SMP, the application of viable pollen from selected donors to un-isolated female strobili has been successful in reducing selfing and pollen contamination3, harmonizing reproductive output differences among orchard’s parents and achieving increased seed yields17.To mitigate the negative effects of seed orchards’ deviation from panmixia, intrusive practices affecting pollination environment such as bloom-delay 5. SSRs have been extensively used to study seed orchards mating patterns20 as they offer increased diagnostic power as compared to the first used allozyme markers22.The availability of highly polymorphic molecular markers such as single sequence repeats (SSRs) and pedigree reconstruction made it feasible to accurately evaluate the effectiveness of seed orchard’s pollination environment manipulation practices through direct assessments of parental reproductive success and pollen contaminationPseudotsuga menziesii) seed orchard7. Throughout the four studied years, this orchard’s pollination environment has been manipulated using combinations of bloom-delay and/or supplemental-mass-pollination under variable seed crop yields. Level of pollen contamination, self-fertilization, as well as parental gametic contribution are reported and compared across years to evaluate the pattern of gene flow and parental reproductive success under different conditions. To our knowledge, this is the first temporal variation study of Douglas-fir seed orchard pollination dynamics while considering both natural and intrusive pollination practices and seed crop sizes variability.Here, we represent a continuation of a multi-year pollination dynamics assessment of a coastal Douglas-fir  produced intermediate contamination rates (13–18%) between those of the concurrent pollination environment manipulation treatments (10%)5 and natural pollination (28%). Furthermore, the difference between 20074 and 20096 could be a reflection of the amount of SMP efforts dedicated to small verses large crops and may highlight SMP ineffectiveness during years with large crops (Table 6 (10–18%) were lower than that observed for natural pollination (28%).The pedigree reconstruction successfully identified the maternal and paternal parents of the fingerprinted seeds and most importantly, identified those pollinated by outside sources , including those from SMP external pollen donors. The contamination rate varied widely among the studied years and ranged between 10% for 2005 where the two intrusive pollination environment manipulation treatments (bloom-delay and SMP) were concurrently implemented23, the true selfing rate should then be estimated after the exclusion of matings resulting from external gene flow. True selfing rates were consistently higher than the overall selfing rates , confirming the role of contamination in under-estimating the effective selfing  in the seed crop (Table 3 and 20074 and 4% for 20096 vs. 6% for 2008)  and 22%  (Table Since every successful contamination event is an outcrossing eventop Table . It is n8) Table . True set) Table .24, the parental gametic contribution fluctuated over the studied years and ranged from 37 to 64% for 20053 and 2008, respectively (Table 3 and 57 (2008), and 45 (2005 and 2009)6 and 59% (2008), respectively  parental inherent fertility and reproductive phenology variation27, (2) uncontrollable external gene flow28, (3) yearly cone/seed crop fluctuations26 , (4) environmental conditions prior and during the pollination season13, and (5) pollination environment manipulation practices29. These factors act individually and in concert creating variable pollination dynamics across the pollination season and among years. The present study presents a complex situation where all these factors are interacting and effectively influencing the pollination dynamics of the studied Douglas-fir seed orchard.Studying pollination dynamics in seed orchards is a daunting task13. Since the orchard population is composed of individuals and their descendants selected from different locations within the orchard’s target planting area , then it is expected that temporal reproductive phenology development variation exists reflecting adaptation to their original location. A protracted pollination season with temporally overlapping smaller reproductively active subpopulations of a small cone crop do not produce enough pollen load to saturate receptive female strobili, hence the high contamination rate. The high external gene flow of 2008 (28%) ought to be contrasted with that of 20053 (10%), another year of small seed crop (3.5 kg)  and SMP treatment produced somewhat contrasting results (13 vs. 18%) (Table 6 (68.8 kg) should have less external gene flow than 20074 (19.3 kg). The obvious explanation for this discrepancy is the ineffectiveness of SMP in larger crops with high within orchard pollen load, a situation that calls for the strategic implementation of this practice (see first-pollination primacy below). The observed external gene flow estimates presented in this study are in line with that reported for another open-pollinated Douglas-fir orchard located in the same area of southern Vancouver Island in which pollen contamination rate of 26% was reported under natural pollination environment8. These external gene flow estimates are common when orchards are located within the target species’ natural range. Higher external gene flow estimates were reported in other orchards with seed crops developed under natural conditions 32 and Scots pine orchard located in northern Sweden (21–36%)28). The negative impact of external gene flow is two-fold: (1) reducing the seed crop’s genetic worth, and (2) maladaptation if the orchard is located outside its seed planning zone33. In the case of the studied orchard, only genetic worth loss is expected and ranged between 7.2 and 17.6% for 2008 and 20074, respectively  − )/) × 100). Finally, it has been argued that external gene flow has a beneficial role through increasing the genetic diversity34; however, this is a tenuous argument considering the extent of genetic diversity in conifers35.External gene flow estimates varied among the studied years with a peak value of 28% in 2008. It should be noted that in 2008 pollination was under natural conditions  and, more importantly, this year has the smallest seed crop among the studied four years (2.4 kg) Table . The com36, (3) SMP effectiveness, and (4) seed crop size . Successful contamination events are in fact outcrossing matings as the pollen donors are not members of the seed orchards population3. Thus, it is of great importance to differentiate between overall and true selfing rates. Overall selfing is the proportion of selfed seed present in the sample of fingerprinted seed, whereas true selfing is the proportion of selfed seed after excluding external gene flow matings. Without exception, the true selfing rate must be higher than the overall selfing rate. Across the four studied years, the true selfing rate was higher than its biased overall counterpart confirming the role of external gene flow on the apparent selfing rate estimation 38. Interestingly, Woods and Heaman38 observed reduced seed set with lower inbreeding levels produced from matings among relatives, indicating that while consanguineous matings do not impact seed set as selfing, they are capable of producing viable seed. These seeds often succeed in producing seedlings in non-competitive environment such as container stock seedlings40, thus resulting in seedlings with reduced survival chance in competitive environments. Among the three years with SMP treatment 6, the observed selfing rate was negatively correlated with the number of pollen donors included in SMP mixes (Table 4: 14%) is associated with the largest number of pollen donors (25) and conversely, the highest selfing rate (20096: 21%) is associated with the fewest pollen donors (14)  rate of gene flow, (2) first-pollination primacy rate of 41, while marinating comparable diversity level as those present in the base population43. Thus, the allelic frequencies of the seed orchard’s parental population  are expected to be maintained in the seed crops, so genetic gain can be captured. This situation can only be attained if the seed orchard population is in Hardy-Weinberg equilibrium, therefore the parental gametic contribution to seed crops is of substantial importance. In many seed orchards, parental fertility variation produced a common scenario where 20% of the parental population contribute 80% of the seed crops24. In the present study, the maternal, paternal, and combined parental gametic contribution differed between sexes and varied among years (Table 4: 19.3 kg) and large (20096: 68:8 kg) seed crops produced a scenario where 80% of the gametes were contributed by approximately 50% of the parents while the small crops (20053: 3.5 kg and 2008: 2.4 kg) resulted in a wide parental contribution difference  rate of 14% under combinations of pollination environment manipulation treatments (bloom-delay and/or SMP), and higher external gene flow (28%) under natural pollination condition. SMP effectiveness was somewhat limited under high within orchard pollen load , multiple application of pollen from low reproductive output parents is recommended for adjusting parental fertility variation. Generally, selfing rate was higher than previously reported and this is attributable to the increased SSRs discrimination power. Comparing the same seed orchard’s performance over multiple years and under different pollination environment treatment provides a general guideline for the role of these treatments on the genetic quality of seed crops. Finally, it should be stated that while the study did not follow the classical factorial experiment where pollination environment manipulation treatments were implemented in all possible combinations, the present study was informative and provided useful information on the impact of pollination environment manipulation on mating system parameters.Pseudotsuga menziesii var. menziesii (Mirb.) Franco) seed orchard established in 1990 provided the material for this investigation. It is located in the Saanich Peninsula, Vancouver Island, British Columbia, Canada  and planted following the permutated neighbourhood design44. The orchard parental population is in constant change as lower breeding value parents are replaced by better ones  over the study period  was employed for DNA amplification for the utilized microsatellite markers . PCR conditions and scoring method are listed in Lai, B. S. et al.5.The bulk seed samples were germinated following stratification45 Table . Germina47 . After pedigree reconstructing of each year’s seed crop, the maternal, paternal, and overall gametic contribution was calculated based on the number of offspring each parent produced as maternal, paternal, and parental (combined).Parentage was assigned to each offspring (seed) in a two-step method using CERVUS 3.0.3Summary of allele frequency analysis for microsatellite marker evaluation


The proton pump dissociates periplasmic AGP-glycomodules that bind Ca2+. Thus, as the immediate source of cytosolic Ca2+, an AGP-Ca2+ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca2+ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca2+ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress–strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca Cell shape is one aspect of the more general problem of differentiation. Consider the primary cell wall, how it grows, and there is evidence enough for the primary wall playing a decisive role in influencing the shape of a cell. Therefore, the primary cell wall must be studied, but how?” Lamport 1963) [963 [1].+, and Ca2+ gradients and how their interaction creates morphogenetic patterns. We therefore approach the formidable problem of plant morphogenesis from an empirical biochemical perspective based on the dynamic role of AGPs  in a novel Hechtian growth oscillator. A cell surface AGP-Ca2+ capacitor is a crucial component that depends on the complex carbohydrate chemistry of classical AGPs [2+ by paired glucuronic carboxyl groups of numerous small glycomodules. Not surprisingly, after their initial discovery as an arabinogalactan polysaccharide [2+ capacitor [2+ and tip-localised AGPs with Hechtian adhesion and rapid tip growth of pollen tubes [In 1952, Turing proposed that “morphogens acting together and diffusing through a tissue is adequate to account for morphogenesis.” His classic paper  “On Growcal AGPs ,8 that cccharide , it has ccharide  to unravapacitor . The rolen tubes .2+. We propose that mechanotransduction generates both gradients, although each has a different biochemical origin; thus, auxin gradients originate from biosynthesis and transport by tissue-specific auxin efflux PIN proteins. On the other hand, cytosolic Ca2+ gradients originate from Ca2+ stored in a cell surface AGP-Ca2+ capacitor [2+ at the periplasmic cell surface seems inescapable to us, the role of AGPs in Ca2+ gradient formation in tissues is less obvious because it depends on a membrane-bound H+-ATPase that dissociates AGP-Ca2+. That shows the close link between auxin and Ca2+ signalling with the primal proton pump. Paired glucuronic carboxyls of AGP glycomodules bind Ca2+ tightly [2+ chelation by dicarboxylic acids). This effectively scavenges less tightly bound Ca2+ of pectin and also free Ca2+ in the transpiration stream. Thus, abundant cell surface AGP-Ca2+ acting as a Ca2+ sink would enable both cells and tissues to compete for Ca2+, so that Ca2+ supply meets demand where it is greatest, such as in meristem primordia. The “choice” of Ca2+ as a universal signalling ion can be ascribed to the size (0.9 Å) of its ionic radius; thus, Ca2+ sheds water more rapidly and is therefore more reactive than other divalent ions—notably Mg2+ [2+.Here, we extrapolate these recent results to morphogenesis and propose two new avenues: Firstly, we propose the avenue of cell walls and mechanoperception involving Hechtian transduction as described in detail here but not considered by Turing; secondly, we propose postulated chemical morphogen gradients  identifiapacitor . AGPs arapacitor ; that exapacitor  and why apacitor . Many AGapacitor ,16,17,18 tightly   cell wall mechanotransduction and the developmental sequence, (2) the embryogenesis of the fertilised egg cell, (3) roots, (4) shoots, and finally (5) the evolution of morphogenesis.2+ capacitor.Most models of morphogenesis, particularly computer-generated models, generally favour a genetic perspective with an emphasis on signalling cascades. However, they ignore two “unknown unknown” missing pieces of the morphogenetic puzzle, identified here as Hechtian mechanotransduction and the AGP-Ca−12 N) forces  forces . 2+ channels and ATPases?How does it distinguish between the huge forces  exerted on the membrane by osmotic pressure and the minute piconewton forces that activate CaHow does it provide a directional signal in response to anisotropic stress? Why does the wall need strong adhesion to the plasma membrane although turgor pressure ensures it? Is the elusive mechanosensor a Boojum or a Snark?However, the Hechtian transduction hypothesis creates three dilemmas: Hechtian adhesion must satisfy several criteria: minimally, it must involve strong, stable and specific interactions with both the cell wall and plasma membrane. Several candidates frequently suggested such as wall-associated kinases and integrins lack hard data and can be eliminated. For example, integrins recognise the RGD motif of the mammalian extracellular matrix but are absent from plants. Other possible candidates include formins and many AGPs anchored securely to the plasma membrane ,28,29,30At-AGP57C (At3g45230) is covalently linked to pectic RG-I homogalacturonan  and is a2+ channels and H+-ATPase; both membrane components are stretch-activated by just a few piconewtons. Wall stress and strain is thus focussed at the molecular level on CaPiezo proteins responsive to mechanical force are well-documented in animal systems . FinallyAlthough osmotic pressure is equally distributed at the plasma membrane, wall pressure exerts an equal and opposite force. However, a direct covalent connection between pectin and the plasma membrane would enable instant transmission of the wall stress–strain status by fibrous macromolecules.Two extreme examples, stomata and pollen tubes, demonstrate the efficacy of Hechtian adhesion: the stomatal guard cell wall elasticity transmits stress while the rapid tip growth of a pollen tube involves wall plasticity; when stretched, it transmits strain arguably via AGP57C  or its h2+ crosslinking forms much stronger gels. A frequently cited pectic paradigm [2+ for crosslinking but ignores the regulation of Ca2+ in muro by AGPs which, with their demonstrated higher affinity for Ca2+, compete for pectic Ca2+. Such competition effectively strips Ca2+ from pectin. The concomitant electrostatic repulsion of negatively charged pectic carboxylates explains the increase in wall plasticity by pectin methylesterase [2+. Significantly, small AGP peptides are upregulated by auxin [The pollen tube tip has the simplest primary cell wall—almost exclusively pectin and with fast tip growth—so is an ideal system to test hypotheses involving the Hechtian transduction of wall stress–strain. Pectin rheology is deceptively simple and depends largely on the degree of methyl esterification of pectic polygalacturonic acid residues, in contrast to AGP glucuronic acid, which is never esterified. Highly methyl-esterified pectins form gels, while de-esterification followed by Caparadigm  assumes esterase . This efesterase  that posby auxin , suggest2+ oscillations and exocytosis (2+ and borate crosslinking of the RG-II pectic component. Crosslinking of the cellulosic component (notably absent from pollen tube tips) may also be regulated by expansin [Driven by turgor pressure, cells expand in a direction orthogonal  to the direction of stress that strains the pectic matrix; the concomitant Hechtian transduction of pectic strain initiates Caocytosis . The strexpansin ,61.2+ in the exocytosis of wall plasticisers as detailed in [2+ by increasing the size of their cell surface AGP-Ca2+ capacitor; this can increase the amplitude of Ca2+ oscillations, thereby enhancing exocytosis.Such complexity explains why the molecular basis of cell wall plasticity remains recalcitrant since first described . “The diailed in  rather tailed in . Cells a2+ channels [+-ATPase [2+ influx as follows: the plasma membrane ATPase [Consider the primary receptors of force transduction. What are they, and how do they propagate tension to the cell interior? Stretch-activated Cachannels  and auxi+-ATPase  control e ATPase ,65 is sie ATPase .2+ of the cell surface capacitor that supplies the Ca2+ channels. The evidence [2+ during the rapid tip growth of pollen tubes.These powerful proton jets dissociate the AGP-Caevidence  for this2+ fluxes translate into morphogenesis depends on the local wall plasticity that determines the direction and magnitude of the wall stress vector; its amplification depends on the size of the AGP-Ca2+ capacitor (How Caapacitor  and its Plant cells exploit a “thermodynamic loophole” based on their ability to trap ionised auxin in the cytosol (pH 7.4) where it cannot freely permeate the plasma membrane. However, in the wall at ~pH 5, auxin is protonated and thus freely permeates the plasma membrane of adjacent cells. Thus, auxin wins the Sysiphean uphill struggle.2+ channel activity and the plasma membrane H+-ATPase  to the outer protoderm (tension). The concomitant rearrangement of auxin efflux proteins in the protoderm under tension results in lateral auxin export from its apex outwards, with consequential slow growth and anisotropy at the torpedo stage that splits the tissue into the two cotyledons [The above description of stress-dependent auxin and Catyledons   with apically polarised PIN4 and PIN7 auxin efflux proteins [The Hechtian growth oscillator generates morphogenetic patterns; arguably, these depend on localised adhesion sites that result in vectorial exocytosis of wall precursors. This includes auxin efflux PIN proteins directed to the sites of the highest strain in expanding cells,  thus ensproteins  highligh2+, thus increasing cytosolic Ca2+ that triggers vectored exocytosis. We infer that Hechtian adhesion and AGPs amplify both the direction and magnitude of stress vectors resulting in growth orientation. Not surprisingly, mutants of Gal31 are lethal [2+ glycomodule [Stress also predicts the plane of cell division marked by the preprophase band of microtubules  as putate lethal , possiblcomodule .2+-signalling was unexpected yet surprisingly close to reality, with AGP-Ca2+ belatedly seen here as the major dynamic source of Ca2+ regulated by auxin. Significantly, transformed plants that overproduce auxin show excessive xylem and phloem development [The morphogenesis of root xylem tissues begins with the specific expression of the JIM13 AGP epitope in a single metaxylem initial just above four central cells of the quiescent centre . The howelopment  and bushelopment .Mechanical stress patterns involved in root morphogenesis have yet to be modelled. Nevertheless, a recent comprehensive experimental approach  correlat2+ [2+ influx” [. Nevertheless, the Cholodny–Went explanation of auxin redistribution remains unquestioned dogma [The presence of the cell wall is essential for gravity perception . These o2+  and the 2+ : “Thus, 2+ ,92,93,94 influx” ,51. Neveed dogma : “The abed dogma . This deThe root–shoot paradox: root tip gravitropism increases auxin at the lower side that decreases growth while less auxin at the upper side increases growth. However, in shoots, a similar auxin distribution in response to gravity reverses the direction of growth. We resolve this paradox by asking: How does auxin inhibit growth at the lower side of the root? Extensin peroxidase  providesWhile the above approach to a complex problem is consistent with Occam’s razor, it contrasts with the complexity of recent papers that deal with the dynamic rearrangement of PIN proteins transporting auxin from both root tip and the shoot via xylem and root epidermis .2+ signalling although [2+ waves,” the major source being the Hechtian oscillator via auxin-activated H+-ATPase dissociation of AGP-Ca2+ that releases Ca2+ to the cytosol via Ca2+ channels. How that translates into increased cell extension in the root tip elongation zone remains a conundrum, as discussed above. Although the prevalent statolith hypothesis is now textbook dogma, the precise molecular details of statolith mechanotransduction remain quite unknown. Mutants that lack amyloplast statoliths do not resolve the problem: while they decrease the gravitational response, they do not eliminate it [+-ATPases [Arabidopsis) and PIN proteins recycle in response to mechanotransduction, thus adding an additional level of complexity.However, no convincing molecular mechanism has been offered for auxin-increased cell extension. Auxin has also not been successfully connected with Caalthough  “auxin sinate it . Finally-ATPases   to redistribute auxin by recycling the auxin efflux PIN proteins. As the root tip derives its auxin from transport through central root tissues and also local biosynthesis  proteins maintained by the cell wall ,108. How2+ by auxin. Thus, auxin efflux PIN proteins transport auxin to target sites, where the activation of H+-ATPase releases Ca2+ from AGP-Ca2+, triggering exocytosis. This paradigm shift can be traced back to the primordial soup and unifies the chemiosmotic proton motive force with auxin and Ca2+ signalling. By analogy with inscriptions on the Rosetta stone, once decoded, these three fundamental transport phenomena translate the same growth message into morphogenesis.Phylogeny and morphogenesis are inextricably linked , and so 4, CO2, and water to yield HCHO [Morphogenesis begins with prebiotic synthesis from CHeld HCHO , a precueld HCHO , a univeeld HCHO  remain tChlamydomonas provides the most striking example of evolutionary conservation and diversification, with a large complement of 182 different proteins related to hydroxyproline-rich extensins [Chlamydomonas wall is devoid of cellulose and consists almost entirely of glycoproteins that self-assemble as a crystal lattice [Emiliana huxleyi of the cretaceous that built the chalk hills and cliffs of the present era.From the cell wall viewpoint, the unicellular flagellate xtensins . The Chl lattice , a remar lattice . The lea3 coccoliths of Coccolithophores represented by E. huxleyi are the probable evolutionary precursor to dynamic cell surface AGP-Ca2+, hence Ca2+ signalling. Algae increased the tensile strength of a primary cell wall prototype by co-opting cellulose, thus enabling turgor pressure and the major evolutionary pathway to metaphytes. Indeed, the composition of algal pectin-rich cellulosic walls is remarkably similar to those defined as primary walls in the cambial tissues of higher plants [2+ signalling. This implies incredible evolutionary conservation and an early origin of the Hechtian pscillator in unicellular Chlorophycean algae such as Closterium [Static cell surface CaCOr plants . That rar plants , we extrosterium  and Peniosterium , as bothSpirogyra [Spirogyra rhizoids, as well as stretch-activated Ca2+ channels, nicely represent the earliest evolutionary progression from marine via freshwater to land plants  that retains the fertilised diploid egg cell until “germination”. Coleochaete cell walls are also significantly enriched in hydroxyproline-rich glycoproteins. Alkaline hydrolysis yields a hydroxyproline glycoside profile  culminated in its complete dominance in higher plants with their increasingly complex division into root, shoots and leaves. Root-like structures appeared as rhizoids in the Bryophytes, but true roots defined by their gravitropic response, endogenous branching, root hairs, and a protective root cap originate from the suspensor hypophysis during angiosperm embryogenesis. While sharing much in common with true shoots, roots clearly lack many shoot characteristics and have therefore been described as “partial shoots”  that evo2+ release and exocytosis. The ultimate reduction of the gametophyte generation to just a few cells, namely pollen and the ovule as an integumented megasporangium, completes this brief summary based on a novel growth oscillator unifying other growth regulators that control CaIon transport dominates this paper, which is greatly influenced (1955–1961) by personal acquaintance (DTAL) with the Cambridge pioneers who included Peter Mitchell (proton pump), David Keilin (cytochrome discovery) and Robert “Robin” Hill Reaction (photolysis of water). The cell wall protein was undoubtedly inspired by Fred Sanger in the adjacent lab , and Joseph E. Varner  on his s


High-speed single-molecule fluorescence microscopy in vivo shows that transcription factors in eukaryotes can act in oligomeric clusters mediated by molecular crowding and intrinsically disordered protein. This finding impacts on the longstanding puzzle of how transcription factors find their gene targets so efficiently in the complex, heterogeneous environment of the cell.Abbreviations CDF - cumulative distribution function; FRAP - fluorescence recovery after photobleaching; GFP - Green fluorescent protein; STORM - stochastic optical reconstruction microscopy; TF - Transcription factor; YFP - Yellow fluorescent protein Cells regulate gene expression through binding of transcription factors (TFs) to promoters to turn gene expression on or off ,2. SimulSaccharomyces cerevisiae (budding yeast). In particular, its glucose sensing pathway presents an experimentally tractable system to study gene regulation. Here, control of gene expression is achieved by TFs which include the Zn finger DNA binding protein Mig1 [GAL genes involved in glucose metabolism [Many of the important features of gene expression control in eukaryotes are exemplified in the model unicellular microorganism ein Mig1  that acttabolism . Mig1 lotabolism , correlatabolism ,22.S. cerevisiae cells, using its glucose sensing pathway as a model for signal transduction, was explored using physics methods which enable the understanding of the processes of life one molecule at a time [In recent investigations from my own group  the spatt a time ,25, emplt a time . The comt a time  in both t a time . These st a time ,30, sucht a time –35, cellt a time –38 and pt a time , along wt a time –43. The t a time –46, and t a time –49.In this Points of View article I discuss further the findings from my team from single-molecule fluorescence microscopy to track functional TFs with very high speed to match typical rates of protein diffusion in live cells and thereby enable “blur-free” observations. We were able to quantify the composition and dynamics of Mig1 under normal and perturbed conditions which affected its state of phosphorylation, and also performed experiments on a protein called Msn2 which functions antagonistically, i.e. instead as an enhancer/activator, for many of the same Mig1 target genes  through S. cerevisiae cells ) using a green fluorescent protein (GFP) reporter for Mig1 integrated into the genome, including mCherry reporter on the RNA polymerase subunit protein Nrd1 to indicate the position of the cell nucleus. Slimfield was optimized for single-molecule detection sensitivity by using an in vitro imaging assay [We used millisecond Slimfield single-molecule fluorescence imaging ,45,51 onae cells ) using ang assay . We alsong assay  and esting assay , but is ng assay . AFM canng assay ), latticng assay ), multi-ng assay .), remotng assay .), and dng assay .).10.108glucose (-/+) we measured cytoplasmic and nuclear Mig1 localization bias respectively, visible in individual cells by our generating rapid microfluidic exchange (a few seconds) of extracellular fluid (Under depleted/elevated extracellular ar fluid ), and rear fluid ). The foar fluid ), with mD as a function of its location in either the cytoplasm, nucleus or translocating across the nuclear envelope, as well as the copy number of Mig1 molecules associated with each subcellular region and in each cell as a whole, indicating ~850–1,300 Mig1 molecules per cell dependent on extracellular glucose. It should be noted that confinement may affect the apparent diffusion coefficient in the small volume of a yeast nucleus if the length the mean square displacement (MSD) of tracked particles is comparable to the diameter of the nucleus, however, if our case only the short length scale MSD regions are considered to determine D.Nanoscale tracking determined the position of tracked Mig1 foci to a lateral precision of 40 nm ,62 couplGSY1 showed colocalization between PP7-GFP expressed off a plasmid and Mig1-mCherry expressed genomically under high glucose conditions. We also observed similar clustering and co-localization to PP7 for the antagonistic TF Msn2. These PP7 co-localization results suggest that clusters both of Mig1 and Msn2 are functionally active in regulating target gene expression of the test target gene GSY1.In control experiments, a modified strain  generateglucose (±), and nuclear foci at glucose (-), were consistent with just a single mobile population whose D of 1–2 μm2/s consistent with earlier observations. However, nuclear foci at glucose (+) indicated a mixture of mobile and immobile components (glucose (+) Mig1 diffusion being impacted by interactions with nuclear structures, similar to that reported for other TFs [lac repressor. Control experiments with Zn finger deletion strains of Mig1 indicated that Mig1 clusters bind to the DNA via their Zn finger motif with direct glucose dependence. At the high laser exceition intensities used for Slimfiled imaging photobleaching is rapid, and so typically a single GFP molecule will photobleach on average after 5–10 consecutive image frame. To account for this we interpolate observed foci brightness values back to the start of each photobleach using an exponential photobleach function. We observed no direct evidence for irreversible photobleaching (i.e. “photoblinking”) with GFP at these intensities, though other fluorescent proteins such as YFP have been known to exhibit such blinking behavior, which if so would need to be further characterized, for example using surface imnmobilized purified YFP in vitro samples. A general compromise here, however, is to confine tracking analysis to typically less than 100 ms of laser exposure so that irreversible photoblinking is more dominant than reversible blinking.Cytoplasmic Mig1 fluorescent foci at mponents ). These ther TFs . Here, tglucose (-) does not exhibit immobility in the nucleus and that Mig1 lacking the Zn finger still accumulates in the nucleus at glucose (+) this suggests that Mig1 localization is driven by changes in Mig1 binding affinity to other proteins, e.g. the general co-repressor complex at the genome [We compared the spatiotemporal dynamics of different Mig1 clusters during translocation by converting trans-nuclear tracks into coordinates parallel and perpendicular to the measured nuclear envelope location, and synchronizing coordinate origins to be at the nuclear envelope crossing point for a given foci track. A heat map of spatial locations of translocating clusters indicated a hotspot of comparable volume to the nuclear pore complexes and accessory structures ,66 Figu). The dwe genome , or outsglucose (-) and (+) with a time constant of just a few seconds but a larger time constant at glucose (+) for nuclear foci of at least ~100s (By modifying the microscope we were able to implement fluorescence recovery after photobleaching (FRAP) to probe nuclear turnover of Mig1, by focusing a separate laser onto just the nucleus, photobleaching this region with a rapid 200 ms pulse, and quantifying any subsequent fluorescence intensity recovery into that region ). We coust ~100s ), with rIn vitro Slimfield imaging of purified Mig1-GFP under identical imaging conditions for live cells similarly indicated monomeric Mig1-GFP foci in addition to a small fraction of brighter foci which were consistent with chance overlap of monomer GFP images. However, addition of a molecular crowding reagent in the form of low molecular weight polyethylene glycol (PEG) at a concentration known to correspond to small molecule “depletion” forces in cells [Native, denaturing gel electrophoresis and western blots on purified extracts from Mig1-GFP cells ) indicatin cells  resultedSecondary structure predictions suggested significant regions of disorder away from the Zn finger binding motif. We measured changes in circular dichroism of the Mig1 fusion construct upon addition of PEG ) in a waOur findings address aspects of functional gene regulation in live cells which have hitherto been unexplored, using biophysical technology that has not been available until recently. The results strongly support a functional link between Mig1 and Msn2 TF clusters and target gene expression; a biological role of multivalent TFs for enhancing intersegmental transfer had been elucidated previously in simulations  but unobS. cerevisiae genome from the 3C model indicates 20–30% are <50 nm apart, small enough to enable different DNA segments to be linked though intersegment transfer by a single cluster [Quantifying nearest-neighbor distances between Mig1 promoter sites in the  cluster ,9, whichOur discovery is, to our knowledge, the first to make a link between predicted disorder and the ability to form oligomeric clusters in TFs. Our findings may potentially offer some insights into addressing the longstanding question of why in general there is so much predicted disorder in eukaryote transcription factors; ~90% of eukaryotic TFs indicate significant proportions of sequences with disordered content . Our fin


Xanthomonas campestris pv. musacearum (Xcm), is a major threat to banana production in east Africa. All cultivated varieties of banana are susceptible to Xcm and only the progenitor species Musa balbisiana was found to be resistant. The molecular basis of susceptibility and resistance of banana genotypes to Xcm is currently unknown. Transcriptome analysis of disease resistant genotype Musa balbisiana and highly susceptible banana cultivar Pisang Awak challenged with Xcm was performed to understand the disease response. The number of differentially expressed genes (DEGs) was higher in Musa balbisiana in comparison to Pisang Awak. Genes associated with response to biotic stress were up-regulated in Musa balbisiana. The DEGs were further mapped to the biotic stress pathways. Our results suggested activation of both PAMP-triggered basal defense and disease resistance (R) protein-mediated defense in Musa balbisiana as early response to Xcm infection. This study reports the first comparative transcriptome profile of the susceptible and resistant genotype of banana during early infection with Xcm and provide insights on the defense mechanism in Musa balbisiana, which can be used for genetic improvement of commonly cultivated banana varieties.Banana Xanthomonas wilt disease, caused by  Xanthomonas campestris pv. musacearum (Xcm), is one of the most devastating disease endangering the livelihood of millions of farmers in east Africa, which is the largest banana-producing and -consuming region in Africa1. The impacts of BXW disease are both rapid and extreme, unlike those of other diseases, which cause gradually increasing losses over years. The disease has caused estimated economic losses of about $2-8 billion over the decade and significant reductions in production have resulted in major price increases1. The disease affects all banana cultivars grown in east Africa2. Only diploid Musa balbisiana, which is a wild type banana native to Asia and one of the progenitors of modern cultivated bananas, was found to be resistant2.Banana Xanthomonas wilt (BXW), caused by the bacterium 3. There is an ongoing project for developing transgenic bananas resistant to BXW using sweet pepper genes4. However, knowledge of resistance mechanism in Musa balbisiana against Xcm can be utilized for developing resistant varieties through cis-genesis using Musa genes associated with defense, or editing of genes related to susceptibility and/or negative regulation of plant immunity. Currently, there is no understanding about the molecular mechanism for disease resistance or susceptibility in response to Xcm infection. Therefore, to obtain insight into the molecular basis of disease resistance, the transcriptome-wide differential gene expression was investigated between the BXW-resistant genotype Musa balbisiana and BXW-susceptible genotype Pisang Awak in response to Xcm. Further the differentially expressed transcripts were mapped to biotic stress pathways using Mapman to identify genes associated with defense mechanism.Resistant cultivars could play an important role in controlling the BXW disease in east Africa, where the consumption of bananas is highest in the world at 220 to 460 kg per person annuallyMusa balbisiana were analyzed for transcriptome comparison.In this study, RNA-Seq data from Xcm challenged and mock-inoculated BXW-susceptible banana genotype Pisang Awak and BXW-resistant genotype Musa acuminata genome version 2. The raw reads were deposited in the National Center for Biotechnology Information Sequence Read Archive under project SRPRJNA401071 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP116676).In total, about 559 million raw reads were generated from sequencing of 30 libraries using Illumina HiSeq™ 2500. The majority of samples had average of 5–10 million reads that uniquely mapped to the reference Readcounts for 35,238 annotated genes were loaded in R program and genes with low expression were filtered out. This filtering step reduced the dataset to 26,676 genes. The DEGs were identified in DESeq2 using filtered readcount data. The data from two time points, 12 h post inoculation (hpi) and 48 hpi, were analyzed and a model was created to identify genes that were responding to the treatment with pathogen and also had an interaction with the genotypes. The design parameter (~genotype + treatment + genotype:treatment) was used for DESeq2. The genes with fold change of greater than 1.5 or <−1.5 were identified.Musa balbisiana in comparison to BXW-susceptible genotype Pisang Awak at both 12 hpi (1749 vs 4) and 48 hpi (245 vs 88)  Fig. –S4. Out ted Fig. .Figure 1Musa balbisiana was considerably higher at 12 hpi in comparison to 48 hpi (1749 vs 245), in contrast a greater number of genes were differentially expressed at 48 hpi than at 12 hpi in Pisang Awak (88 vs 4)  Fig. . In Musahpi Fig. .Several DEGs were observed due to genotype:treatment interactions , was activated in Pisang Awak at 12 hpi  proteins were up-regulated and also many PR genes were suppressed in response to pathogen interaction with Musa balbisiana as early response to pathogen at 12 hpi  gene in leucine-rich repeat (LRR) family protein and putative disease resistance protein RPM1 were activated in Musa balbisiana at 12 hpi  was suppressed in BXW-resistant Musa balbisiana at 12 hpi , bHLH and C2H2 were also up-regulated in Musa balbisiana at 48 hpi. The MYB transcription factor was also activated in Pisang Awak at both 12 hpi and 48 hpi  to Xcm.Transcripts involved in redox regulation like glutaredoxins, peroxiredoxin, thioredoxins, ascorbate, glutathione, catalases and heme family were up-regulated in Musa balbisiana. Abscisic acid (ABA) metabolism was activated in Musa balbisiana at both 12 and 48 hpi  using a set of 30 annotated genes differentially expressed in response to Xcm infection in The comparison of RNA-Seq and qRT-PCR showed similar trends of expression for candidate transcripts but difference in magnitude Fig. . PearsonMusa balbisiana has resistance to BXW, whereas all cultivated banana varieties including commercial Cavendish varieties are susceptible. Currently, molecular basis of resistance and susceptibility of banana to Xcm is unknown. Therefore, transcriptome profiles of BXW-resistant and BXW-susceptible banana genotypes challenged with Xcm were compared to better understand molecular mechanism of resistance or susceptibility of banana upon bacterial infection. In presence of Xcm, resistant genotype showed higher number of DEGs at 12 hpi in comparison to 48 hpi, suggesting that defense pathways were activated by pathogen infection as early as 12 hpi.BXW is the most important production constraint of banana affecting livelihood of millions of people in east Africa. The wild type banana progenitor Plants defend themselves against pathogens by activating defense mechanisms, including hypersensitive response (HR), induction of genes encoding PR, antimicrobial peptides, generation of reactive oxygen species (ROS), and enforcement of the cell wall.Musa balbisiana after artificial inoculation with Xcm suggested its involvement in plant defense and suppression of bacterial population like action of other AMPs. Vicilin-like AMPs are plant derived α-amylase inhibitors having antibacterial and antifungal activity5.Up-regulation of vicilin-like AMP in 6. Suppression of E3 ubiquitin ligases PUB22 and PUB23 in BXW-resistant genotype Musa balbisiana in response to Xcm was in agreement with previous report demonstrating role of E3 ubiquitin ligases  as negative regulators of PTI in Arabidopsis6.Once plants recognize pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs), series of reactions are activated leading to PAMP-triggered immunity (PTI) as first line of plant immunity or basal defense. E3 ubiquitin ligase plays important role in PTI7. OsWAK14, which is similar to WAK2, was reported to be positive regulator of blast resistance in rice8. LRR-RKs recognizes the pathogen effectors and induces innate immunity. Receptor kinase DUF26 also might have been involved in induction of disease resistance as previously reported that cysteine-rich receptor-like kinases (CRKs) containing DUF26 motif in their extracellular domains are involved in stress resistance9. The previous report demonstrating that AtLRK10L1.2, Arabidopsis ortholog of wheat LRK10 is involved in ABA-mediated signaling10, suggested that up-regulation of LRK10 like receptor kinase in Musa balbisiana should have activated the ABA signaling pathway.Receptor-like kinases act as PRRs, which recognize pathogens as the first layer of inducible defense. Our results showed activation of several receptor like kinases and receptor kinases in BXW-resistant genotype, indicating their role in plant defense Table . WAKs ar11. In this study, activation of MAPK in Musa balbisiana indicated that up-regulation of MAPK cascades in the BXW-resistant genotype might have signaled early defense responses, including generation of ROS and HR leading to program cell death.PTI involves activation of oxidative burst, MAPK activity and transcription factors, preventing colonization of pathogen in host cells and providing disease resistance. MAPK cascades are major plant plasma membrane-localized PRRs, signaling early basal defense responses against pathogen infection12. In this study, differential expression of several genes associated with redox state was observed in BXW-resistant genotype Musa balbisiana at 12 hpi as early response to Xcm infection, whereas no change in redox state was detected in BXW-susceptible genotype Pisang Awak at 12 hpi  during HR in response to powdery mildew16.Our results demonstrated activation of polyamine oxidases and peroxidases involved in ROSMusa balbisiana at 12 hpi, indicating cell wall reinforcement  and ethylene (ET)31, which was also noticed in Musa balbisiana at 12 hpi in response to pathogen attack  transcription factors were suppressed in the BXW-resistant genotype indicating their involvement as negative regulator  pathway in Musa balbisiana at 12 hpi.The phytohormones SA, JA and ET play key roles in defense responses against pathogens41. Resistance proteins protect the plant against pathogens after recognition of virulence factor, however, in the absence of specific recognition of virulence factors, basal defense response occurs through PAMPs. Similar to our studies, it has been reported overlapping of the PAMP-triggered defense with R-protein-mediated defense42.A second line of defense is Effector-Triggered Immunity (ETI), which involves R-gene families. The R-genes interactwith virulence factors of pathogens and trigger defense response characterized by rapid calcium fluxes, oxidative burst, transcriptional reprogramming within and around the infection sites and localized programmed cell death, which leads  to suppression of pathogen growthMusa balbisiana at 12 hpi inducing immune response providing resistance against pathogen  genes, MLO-like protein 13 was found to be suppressed in Musa balbisiana in response to Xcm inoculation indicates their role in innate immune responses like HR and systemic acquired resistance (SAR) in plants against pathogen infection. Osmotin-like protein, a PR5, is reported to be involved in plant defense responses to several pathogens and abiotic stresses46. Transgenic sesame overexpressing osmotin-like PR gene demonstrated resistance against Macrophomena phaseolina infection by activating JA/ET and SA pathways46.Induction of PR genes in BXW-resistant genotype 47. It has been reported that differences between susceptibility and resistance are associated with differences in the timing and magnitude of the induced response rather than just with the expression of various genes48. Further investigation is required to understand the function of GER1 in banana.Germin-like protein (GER1 or GLP1) was activated in both BXW-susceptible and BXW-resistant genotypes at 12 hpi and 48 hpi, respectively. Germin-like protein are reported to be increased in several plants after pathogen infection and involved in plant defenseMusa balbisiana in response to pathogen at both 12 hpi and 48 hpi, whereas proteolysis was also induced at 48 hpi in Pisang Awak  and highly susceptible banana cultivar Pisang Awak  were obtained from in vitro collection at IITA-Kenya. The in vitro plantlets were micropropagated in tissue culture.The BXW disease resistant genotype 8 cfu/ml with sterile water. Fresh inoculum was used for all the experiments in order to have high virulent potential of the pathogen.Pure culture of Ugandan isolate of Xcm isolate collected from Pathology Laboratory, IITA was maintained on YTSA medium  at 4 °C. A single bacterial colony was inoculated into 25 ml of YTS medium  and cultured at 28 °C with shaking at 150 rpm for 48 h. The bacterial culture was centrifuged at 5000 rpm for 5 min and pellet was re-suspended in sterile double distilled water. The optical density (OD 600 nm) of the bacterial suspension was checked and bacterial concentration was adjusted to 10in vitro rooted plantlets with 3–4 leaves were artificially inoculated with fresh culture of Xcm. About 100 μl of bacterial suspension (108 cfu ml−1) was artificially injected using insulin syringe into the midrib of the second fully open leaf. This method of artificial inoculation is similar to natural infection through injury by contaminated cutting tools, commonly practiced by farmers. Previous study in our laboratory showed development of BXW symptoms and complete wilting of Pisang Awak plantlets artificially inoculated with Xcm54. The inoculated leaf of M. balbisiana also showed necrotic and chlorotic patches due to hypersensitive response, but these symptoms did not progress and the plants were subsequently healthy54.One month-old Additional plantlets were mock inoculated with sterile water as control in order to nullify the effect of wounding through injection.55. Three biological and three technical replicates were used for each time point.The inoculated leaf samples were collected at 0, 12 hpi, 48 hpi. These time points were selected as the expression of defense genes during early infection with Xcm was observed to be high at 12 hpi and then decreasing at 48 hpiPisang Awak inoculated with bacterial culture at 0, 12 hpi, 48 hpiMusa balbisiana inoculated with bacteria at 0, 12 hpi, 48 hpiPisang Awak mock inoculated with water at 12 hpi, 48 hpiMusa balbisiana mock inoculated with water at 12 hpi, 48 hpiThe samples were:Total RNA was extracted from 100 mg leaf samples using the RNeasy plant mini kit  and treated with DNase . The quality of RNA was assessed using denaturing agarose gel stained with gelred  and quantified using a Nanodrop 2000 .A total of 30 cDNA libraries were prepared in each of the two treatments [three biological replicates at each of the three time points  per genotype inoculated with bacterial culture and two time points  inoculated with sterile water] using Illumina TruSeq RNA Sample Preparation Kit v2. Poly (A) mRNA was obtained from 1 µg of the total RNA using poly-T oligos attached to magnetic beads. The purified mRNA was fragmented and used to synthesize first strand cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and random primers. Second strand cDNA was synthesized from the resultant first strand and this was end-repaired by adding ‘A’ (A-tailing) at the 3′ ends to allow the ligation of indexed adaptors. The adaptor indexes were used in such a way that it allowed for multiplexing in sequencing. The libraries were enriched through twelve cycles of PCR amplifications. Quantitative and qualitative assessments of the libraries were done using QuBit DNA assay (Thermo Fisher Scientific) as well as with Agilent DNA 1000 assay  on an Agilent 2100 bioanalyzer. All the assays on quality and quantity were performed according to the respective manufacturer’s protocols.The cDNA library was sequenced pair-ends wise using the Illumina HiSeq™ 2500, and 100-bp reads were generated. The sequencing was performed by Center for Genomics and Systems Biology, New York University.Musa acuminata genome using Hisat256. The reference genome for Musa acuminata DH Pahang annotation version 2 and annotations were downloaded from Banana Genome Hub57 (http://banana-genome-hub.southgreen.fr/download). This genome sequence is considered to be the reference genome for Musa species due to its completeness and quality of annotation58. The quality of annotation has recently been quantified through a BUSCO analysis, which scores the genome of Musa acuminata DH Pahang at 96.5% and the genome of Musa balbisiana Pisang Klutuk Wulung at 66.5%59. This genome sequence has been extensively used in other transcriptome studies on Musa including genotypes having B-genome60.The reads were trimmed for quality using Trimmomatic and aligned against the reference Musa acuminata DH Pahang and Musa balbisiana Pisang Klutuk Wulung genomes were downloaded and the reciprocal best BLAST hits were obtained using a perl script. The gene identifiers (IDs) from Musa balbisiana Pisang Klutuk Wulung are reported in all tables based on the results of the reciprocal best BLAST search. The best BLAST hit is reported for the genes where the reciprocal best BLAST search hit was not available and it is denoted with the symbol “*” after the gene identifier.The protein sequences from the HTSeq-count was used to count the number of reads that map to the different annotated genes. Readcounts for annotated genes were loaded in R and genes with low expression were filtered out. The genes were removed from the dataset if none of its groups had a median of 10 reads mapping to the genome. The filtered readcount data was used as input for DESeq2 to determine differentially expressed genes. The genes with fold change of greater than 1.5 or <−1.5 and also an adjusted p-value less FDR corrected p-value of 0.1 were identified.The change in gene expression due to bacterial inoculation was calculated for both genotypes at each time point by comparing them with mock-inoculated plants of the same genotype using the contrasts function in DESeq2. Also the interaction between genotypes and pathogen i.e. genotypes:treatment interaction was recorded.61. GOStats has a functionality that allows individuals to create their own GO annotation database for species that are currently not supported in the annotation databases. Using the Banana Genome Hub Gene-to-GO association a Gene Set Enrichment dataset was created and used for identifying GO-terms that were over-represented in a given gene list. The function has been enhanced to provide the FDR corrected p-values. All go-terms were adjusted for p-value of less than 0.05.A Gene Ontology (GO) Enrichment Analysis of differentially expressed genes was performed using GO-terms provided by the Banana Genome Hub and the GOStats package in R62 version 3.5.1R2. The mapping to the Mapman functional categories (BINs) was done for all the protein sequences from the Musa acuminata version 2 genome. The protein sequences were uploaded to the Mercator tool63. The pathway analysis used the overall metabolic pathway and the biotic stress pathways for this study.The DEGs were mapped to different pathways using the MapmanTo verify the results of RNA-Seq analysis, qRT-PCR analysis was performed with 30 selected DEGs using gene specific primers  of Xcm. The plantlets mock inoculated with sterile water were used as control. The inoculated leaf samples were collected at 0, 12 hpi, 48 hpi and RNA were isolated using the RNeasy plant mini kit  and treated with DNase . First strand cDNA was synthesized using reverse transcriptase of the Maxima H Minus First Strand cDNA synthesis kit with oligo DT primers (Thermo scientific). The primer specificity for each gene was confirmed by PCR and melting curve in qRT-PCR.The samples for qRT-PCR were independently prepared as described above for RNAseq. One month-old Musa25S ribosomal gene as reference. Mock inoculated control plant was used as calibrator to calculate log2 fold change of target gene using the ΔΔCt method64.The qRT-PCR was performed using Maxima SYBR green/ROX PCR kit (Thermo Scientific) on 7900 Real Time PCR System . The experiment was set up using three biological and three technical replicates. Relative expression data were normalized using the The means and standard error were calculated for three replicates per experiment using Minitab 16 statistical software, 2012.The log2 fold change by RNA-Seq and qRT-PCR analysis were compared using Pearson correlation analysis in Minitab 16 statistical software.Supplementary Tables


Melanotic Xp11 translocation renal cancer (TRC) is a newly described exceedingly rare tumor, and its characterization remains controversial. This study aimed to describe a case of distinctive melanotic Xp11 TRC and to elucidate its clinicopathological and molecular genetic features.TFE3 rearrangement.A 44-year-old Chinese female presented with a left renal mass. Abdominal ultrasonography and computed tomography (CT) scans revealed a 4.5 cm × 4.0 cm mass in the left kidney. Grossly, the well-demarcated mass was black with moderately firm consistency. Microscopic examination indicated that the tumor was characterized by the presence of nests and cords of polygonal cells with clear and granular eosinophilic cytoplasm, central round to oval nuclei and occasional nucleoli. Intracytoplasmic melanin was observed in approximately 45% of tumor cells. Uniquely, the tumor presented with intranuclear eosinophilic pseudoinclusions and thick-walled stromal blood vessels. IHC showed that tumor cells were diffusely positive for TFE3 and exhibited patchy and weak HMB45 staining. FISH confirmed the presence of This case is the twentieth published case of melanotic Xp11 TRC. Moreover, the present patient had a favorable prognosis given that she was disease free at her 113-month postoperative follow-up. Our case adds to the small body of literature on these exceptionally rare tumors and widens their clinicopathological spectrum. TFE3) gene. Since it was first described by Argani et al. in 2009, only 19 cases of melanotic Xp11 TRC have been documented in English-language publications  is a newly defined entity that is characterized by sheets and nests of epithelioid neoplastic cells with clear to finely granular eosinophilic cytoplasm and intracytoplasmic melanin; immunohistochemical positivity for melanocytic markers (such as HMB45 and Melan A) but negativity for epithelial and muscular markers; and rearrangement of the transcription factor enhancer 3 , suggesting that this tumor has an indolent clinical course. Second, occasional intranuclear eosinophilic cytoplasmic pseudoinclusions of neoplastic cells were detected in this patient’s tumor. Finally, massive thick-walled blood vessels were observed. In contrast to those found in conventional angiomyolipoma (AML), thick-walled vessels in the current lesion exhibited normal elastic fibers and focal eccentric hyalinization. To our knowledge, these three unique features of our case have not been described in the 19 previously reported cases of melanotic Xp11 TRC;therefore, this case widens this entity’s clinicopathological spectrum.Given the location and pathological appearance of the type of tumor observed in this case, the main differential diagnoses considered should include Xp11 translocation renal cell carcinoma, malignant melanoma, and PEComa, including AML and its epithelioid variant. In fact, the initial clinical and pathological diagnosis for our case was Xp11 translocation renal cell carcinoma due to the tumor’s renal location and histological features of sheets and nests of epithelioid cells with clear to finely granular eosinophilic cytoplasm and intervening delicate fibrovascular septa. However, immunohistochemical negativity for epithelial markers does not exclude Xp11 translocation renal cell carcinoma. Xp11 translocation cell carcinomas are positive for renal tubular markers  at varying levels , 15. In We report a case of melanotic Xp11 TRC with unique histologic features in a 44-year-old Chinese female. This case adds to the small body of literature on these exceptionally rare tumors and widens their clinicopathological spectrum. Further studies are required to determine the precise clinicopathologic and genetic features of such tumors.