Genomic characterisation and dissection of the onset of resistance to acetyl CoA carboxylase-inhibiting herbicides in a large collection of Digitaria insularis from Brazil.

An in-depth genotypic characterisation of a diverse collection of Digitaria insularis was undertaken to explore the neutral genetic variation across the natural expansion range of this weed species in Brazil. With the exception of Minas Gerais, populations from all other states showed high estimates of expected heterozygosity (HE > 0.60) and genetic diversity. There was a lack of population structure based on geographic origin and a low population differentiation between populations across the landscape as evidenced by average Fst value of 0.02. On combining haloxyfop [acetyl CoA carboxylase (ACCase)-inhibiting herbicide] efficacy data with neutral genetic variation, we found evidence of presence of two scenarios of resistance evolution in this weed species. Whilst populations originating from north-eastern region demonstrated an active role of gene flow, populations from the mid-western region displayed multiple, independent resistance evolution as the major evolutionary mechanism. A target-site mutation (Trp2027Cys) in the ACCase gene, observed in less than 1% of resistant populations, could not explain the reduced sensitivity of 15% of the populations to haloxyfop. The genetic architecture of resistance to ACCase-inhibiting herbicides was dissected using a genome wide association study (GWAS) approach. GWAS revealed association of three SNPs with reduced sensitivity to haloxyfop and clethodim. In silico analysis of these SNPs revealed important non-target site genes belonging to families involved in herbicide detoxification, including UDPGT91C1 and GT2, and genes involved in vacuolar sequestration-based degradation pathway. Exploration of five genomic prediction models revealed that the highest prediction power (≥0.80) was achieved with the models Bayes A and RKHS, incorporating SNPs with additive effects and epistatic interactions, respectively.

The floral development of the allotetraploid Coffea arabica L. correlates with a small RNA dynamic reprogramming.

Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage-specific manner. Members of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage-specific manner, related to modulated hormonal responses and transcription factor expression.

LysM receptors in Coffea arabica: Identification, characterization, and gene expression in response to Hemileia vastatrix.

Pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) localized on the host plasma membrane. These receptors activate a broad-spectrum and durable defense, which are desired characteristics for disease resistance in plant breeding programs. In this study, candidate sequences for PRRs with lysin motifs (LysM) were investigated in the Coffea arabica genome. For this, approaches based on the principle of sequence similarity, conservation of motifs and domains, phylogenetic analysis, and modulation of gene expression in response to Hemileia vastatrix were used. The candidate sequences for PRRs in C. arabica (Ca1-LYP, Ca2-LYP, Ca1-CERK1, Ca2-CERK1, Ca-LYK4, Ca1-LYK5 and Ca2-LYK5) showed high similarity with the reference PRRs used: Os-CEBiP, At-CERK1, At-LYK4 and At-LYK5. Moreover, the ectodomains of these sequences showed high identity or similarity with the reference sequences, indicating structural and functional conservation. The studied sequences are also phylogenetically related to the reference PRRs described in Arabidopsis, rice, and other plant species. All candidates for receptors had their expression induced after the inoculation with H. vastatrix, since the first time of sampling at 6 hours post-inoculation (hpi). At 24 hpi, there was a significant increase in expression, for most of the receptors evaluated, and at 48 hpi, a suppression. The results showed that the candidate sequences for PRRs in the C. arabica genome display high homology with fungal PRRs already described in the literature. Besides, they respond to pathogen inoculation and seem to be involved in the perception or signaling of fungal chitin, acting as receptors or co-receptors of this molecule. These findings represent an advance in the understanding of the basal immunity of this species.

Sensitivity assessment and SDHC-I86F mutation frequency of Phakopsora pachyrhizi populations to benzovindiflupyr and fluxapyroxad fungicides from 2015 to 2019 in Brazil.

BACKGROUND: Fungicides of the succinate dehydrogenase inhibitors (SDHIs) group have been used in soybean to control Asian soybean rust (ASR) caused by Phakopsora pachyrhizi. Fungal populations with less sensitivity to SDHI fungicides have been reported since 2015. RESULTS: In this study, fungal sensitivity to benzovindiflupyr (BZV) and fluxapyroxad (FXD) was assessed using a total of 770 P. pachyrhizi populations sampled over four soybean growing seasons. Cross-resistance, intrinsic activity, and frequency of SDHC-I86F mutation were also analyzed. The average effective concentration to inhibit 50% (EC50 ) and SDHC-I86F frequency increased over the 2015/2016, 2016/2017, 2017/2018 and 2018/2019 soybean-seasons. Fourteen P. pachyrhizi populations had the EC50 value above 10 mg L-1 for both carboxamides. No difference was found in intrinsic active to BZV and FXD fungicides for sensitive P. pachyrhizi populations. For P. pachyrhizi classified as less sensitive BZV showed the highest fungitoxicity effect. High frequency of the C-I86F mutation was observed in samples collected in volunteer soybean plants. The maximum frequency of SDHC-I86F mutation in the population was 50% and resulting in ASR populations with low sensitivity to SDHIs. A low correlation between bioassay and SDHC-I86F mutation was observed possible due to the dikaryotic nature of rust fungi or other mutations in the other succinate dehydrogenase subunits. CONCLUSION: The present work provides an overview of a large sampling size of P. pachyrhizi populations and their performance over the four crop seasons. The high frequency of SDHC-I86F mutation and low sensitivity to SDHIs are widely distributed in the main soybean growing regions in Brazil and present in volunteer plants in the soybean-free period. Further detailed studies are needed to identify novel point mutations affecting the effectiveness of SDHIs. © 2021 Society of Chemical Industry.

Reproductive phasiRNA loci and DICER-LIKE5, but not microRNA loci, diversified in monocotyledonous plants.

In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3'-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants.

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

Construction and characterization of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare).

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Correction to: small RNA discovery in the interaction between barley and the powdery mildew pathogen.

Following the publication of the original article [1], the authors noted several typesetting errors which are noted in this Correction article.

Genome-Wide Reinforcement of DNA Methylation Occurs during Somatic Embryogenesis in Soybean.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Small RNA discovery in the interaction between barley and the powdery mildew pathogen.

BACKGROUND: Plants encounter pathogenic and non-pathogenic microorganisms on a nearly constant basis. Small RNAs such as siRNAs and miRNAs/milRNAs influence pathogen virulence and host defense responses. We exploited the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare) to explore fungal and plant sRNAs expressed during Bgh infection of barley leaf epidermal cells. RESULTS: RNA was isolated from four fast-neutron immune-signaling mutants and their progenitor over a time course representing key stages of Bgh infection, including appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. The Cereal Introduction (CI) 16151 progenitor carries the resistance allele Mla6, while Bgh isolate 5874 harbors the AVRa6 avirulence effector, resulting in an incompatible interaction. Parallel Analysis of RNA Ends (PARE) was used to verify sRNAs with likely transcript targets in both barley and Bgh. Bgh sRNAs are predicted to regulate effectors, metabolic genes, and translation-related genes. Barley sRNAs are predicted to influence the accumulation of transcripts that encode auxin response factors, NAC transcription factors, homeodomain transcription factors, and several splicing factors. We also identified phasing small interfering RNAs (phasiRNAs) in barley that overlap transcripts that encode receptor-like kinases (RLKs) and nucleotide-binding, leucine-rich domain proteins (NLRs). CONCLUSIONS: These data suggest that Bgh sRNAs regulate gene expression in metabolism, translation-related, and pathogen effectors. PARE-validated targets of predicted Bgh milRNAs include both EKA (effectors homologous to AVRk1 and AVRa10) and CSEP (candidate secreted effector protein) families. We also identified barley phasiRNAs and miRNAs in response to Bgh infection. These include phasiRNA loci that overlap with a significant proportion of receptor-like kinases, suggesting an additional sRNA control mechanism may be active in barley leaves as opposed to predominant R-gene phasiRNA overlap in many eudicots. In addition, we identified conserved miRNAs, novel miRNA candidates, and barley genome mapped sRNAs that have PARE validated transcript targets in barley. The miRNA target transcripts are enriched in transcription factors, signaling-related proteins, and photosynthesis-related proteins. Together these results suggest both barley and Bgh control metabolism and infection-related responses via the specific accumulation and targeting of genes via sRNAs.

Genome sequencing and transcript analysis of Hemileia vastatrix reveal expression dynamics of candidate effectors dependent on host compatibility.

Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Current knowledge of the H. vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. This resulted in a 547 Mb genome of H. vastatrix race XXXIII (Hv33), with 13,364 predicted genes that encode 13,034 putative proteins with transcriptomic support. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. From this putative secretome, 111 proteins were identified as candidate effectors (EHv33) unique to H. vastatrix, and a subset consisting of 17 EHv33 genes was selected for a temporal gene expression analysis during infection. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Another nine genes were significantly induced after haustorium formation in the compatible interaction. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process.

Reproductive phasiRNAs in grasses are compositionally distinct from other classes of small RNAs.

Little is known about the characteristics and function of reproductive phased, secondary, small interfering RNAs (phasiRNAs) in the Poaceae, despite the availability of significant genomic resources, experimental data, and a growing number of computational tools. We utilized machine-learning methods to identify sequence-based and positional features that distinguish phasiRNAs in rice and maize from other small RNAs (sRNAs). We developed Random Forest classifiers that can distinguish reproductive phasiRNAs from other sRNAs in complex sets of sequencing data, utilizing sequence-based (k-mers) and features describing position-specific sequence biases. The classification performance attained is > 80% in accuracy, sensitivity, specificity, and positive predicted value. Feature selection identified important features in both ends of phasiRNAs. We demonstrated that phasiRNAs have strand specificity and position-specific nucleotide biases potentially influencing AGO sorting; we also predicted targets to infer functions of phasiRNAs, and computationally assessed their sequence characteristics relative to other sRNAs. Our results demonstrate that machine-learning methods effectively identify phasiRNAs despite the lack of characteristic features typically present in precursor loci of other small RNAs, such as sequence conservation or structural motifs. The 5'-end features we identified provide insights into AGO-phasiRNA interactions. We describe a hypothetical model of competition for AGO loading between phasiRNAs of different nucleotide compositions.

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These "phasiRNAs" are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.

Cis-directed cleavage and nonstoichiometric abundances of 21-nucleotide reproductive phased small interfering RNAs in grasses.

Post-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNAs that are processed into 21- and 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275. We characterized these 'reproductive phasiRNAs' from rice (Oryza sativa) panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions. We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production. We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived 'top' strand phasiRNAs are consistently higher in abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.

The asparagus genome sheds light on the origin and evolution of a young Y chromosome.

Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.

Characterization of Plant Small RNAs by Next Generation Sequencing.

Plant small RNAs are ∼20 to 24 nucleotide noncoding RNAs that typically have repressive regulatory roles in gene expression, functioning at the transcriptional or post-transcriptional level. This influence on regulation of developmental and physiological processes has direct effects on phenotype. High-throughput sequencing technologies have enabled the sequencing of millions of small RNAs. Along with decreased sequencing costs, recent improvements in small RNA library construction have facilitated the ability to use minimal amounts of input RNA for analysis. This unit describes steps to isolate total RNA from limited amounts of plant tissue to construct small RNA libraries and perform small RNA data processing. © 2017 by John Wiley & Sons, Inc.

PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice.

Phased small-interfering RNAs (phasiRNAs) are a special class of small RNAs, which are generated in 21- or 24-nt intervals from transcripts of precursor RNAs. Although phasiRNAs have been found in a range of organisms, their biological functions in plants have yet to be uncovered. Here we show that phasiRNAs generated by the photopheriod-sensetive genic male sterility 1 (Pms1) locus were associated with photoperiod-sensitive male sterility (PSMS) in rice, a germplasm that started the two-line hybrid rice breeding. The Pms1 locus encodes a long-noncoding RNA PMS1T that was preferentially expressed in young panicles. PMS1T was targeted by miR2118 to produce 21-nt phasiRNAs that preferentially accumulated in the PSMS line under long-day conditions. A single nucleotide polymorphism in PMS1T nearby the miR2118 recognition site was critical for fertility change, likely leading to differential accumulation of the phasiRNAs. This result suggested possible roles of phasiRNAs in reproductive development of rice, demonstrating the potential importance of this RNA class as regulators in biological processes.

MicroRNA Maturation and MicroRNA Target Gene Expression Regulation Are Severely Disrupted in Soybean dicer-like1 Double Mutants.

Small nonprotein-coding microRNAs (miRNAs) are present in most eukaryotes and are central effectors of RNA silencing-mediated mechanisms for gene expression regulation. In plants, DICER-LIKE1 (DCL1) is the founding member of a highly conserved family of RNase III-like endonucleases that function as core machinery proteins to process hairpin-like precursor transcripts into mature miRNAs, small regulatory RNAs, 21-22 nucleotides in length. Zinc finger nucleases (ZFNs) were used to generate single and double-mutants of putative soybean DCL1 homologs, DCL1a and DCL1b, to confirm their functional role(s) in the soybean miRNA pathway. Neither DCL1 single mutant, dcl1a or dcl1b plants, exhibited a pronounced morphological or molecular phenotype. However, the dcl1a/dcl1b double mutant expressed a strong morphological phenotype, characterized by reduced seed size and aborted seedling development, in addition to defective miRNA precursor transcript processing efficiency and deregulated miRNA target gene expression. Together, these findings indicate that the two soybean DCL1 paralogs, DCL1a and DCL1b, largely play functionally redundant roles in the miRNA pathway and are essential for normal plant development.

Transcriptomics of the rice blast fungus Magnaporthe oryzae in response to the bacterial antagonist Lysobacter enzymogenes reveals candidate fungal defense response genes.

Plants and animals have evolved a first line of defense response to pathogens called innate or basal immunity. While basal defenses in these organisms are well studied, there is almost a complete lack of understanding of such systems in fungal species, and more specifically, how they are able to detect and mount a defense response upon pathogen attack. Hence, the goal of the present study was to understand how fungi respond to biotic stress by assessing the transcriptional profile of the rice blast pathogen, Magnaporthe oryzae, when challenged with the bacterial antagonist Lysobacter enzymogenes. Based on microscopic observations of interactions between M. oryzae and wild-type L. enzymogenes strain C3, we selected early and intermediate stages represented by time-points of 3 and 9 hours post-inoculation, respectively, to evaluate the fungal transcriptome using RNA-seq. For comparative purposes, we also challenged the fungus with L. enzymogenes mutant strain DCA, previously demonstrated to be devoid of antifungal activity. A comparison of transcriptional data from fungal interactions with the wild-type bacterial strain C3 and the mutant strain DCA revealed 463 fungal genes that were down-regulated during attack by C3; of these genes, 100 were also found to be up-regulated during the interaction with DCA. Functional categorization of genes in this suite included those with roles in carbohydrate metabolism, cellular transport and stress response. One gene in this suite belongs to the CFEM-domain class of fungal proteins. Another CFEM class protein called PTH11 has been previously characterized, and we found that a deletion in this gene caused advanced lesion development by C3 compared to its growth on the wild-type fungus. We discuss the characterization of this suite of 100 genes with respect to their role in the fungal defense response.

Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: The rice blast fungus, Magnaporthe oryzae is a destructive pathogen of rice and other related crops, causing significant yield losses worldwide. Endogenous small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs) are critical components of gene regulation in many eukaryotic organisms. Recently several new species of sRNAs have been identified in fungi. This fact along with the availability of genome sequence makes M. oryzae a compelling target for sRNA profiling. We have examined sRNA species and their biosynthetic genes in M. oryzae, and the degree to which these elements regulate fungal stress responses. To this end, we have characterized sRNAs under different physiological stress conditions, which had not yet been examined in this fungus. RESULTS: The resulting libraries are composed of more than 37 million total genome matched reads mapping to intergenic regions, coding sequences, retrotransposons, inverted, tandem, and other repeated regions of the genome with more than half of the small RNAs arising from intergenic regions. The 24 nucleotide (nt) size class of sRNAs was predominant. A comparison to transcriptional data of M. oryzae undergoing the same physiological stresses indicates that sRNAs play a role in transcriptional regulation for a small subset of genes. Support for this idea comes from generation and characterization of mutants putatively involved in sRNAs biogenesis; our results indicate that the deletion of Dicer-like genes and an RNA-Dependent RNA Polymerase gene increases the transcriptional regulation of this subset of genes, including one involved in virulence. CONCLUSIONS: Various physiological stressors and in planta conditions alter the small RNA profile of the rice blast fungus. Characterization of sRNA biosynthetic mutants helps to clarify the role of sRNAs in transcriptional control.