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The role of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily proteins in the innate immune responses of mammals is well characterized. However, the biological function of CAP superfamily proteins in plant-microbe interactions is poorly understood. We used proteomics and transcriptome analyses to dissect the apoplastic effectors secreted by the oomycete Phytophthora sojae during early infection of soybean leaves. By transiently expressing these effectors in Nicotiana benthamiana, we identified PsCAP1, a novel type of secreted CAP protein that triggers immune responses in multiple solanaceous plants including N. benthamiana. This secreted CAP protein is conserved among oomycetes, and multiple PsCAP1 homologs can be recognized by N. benthamiana. PsCAP1-triggered immune responses depend on the N-terminal immunogenic fragment (aa 27-151). Pretreatment of N. benthamiana with PsCAP1 or the immunogenic fragment increases plant resistance against Phytophthora. The recognition of PsCAP1 and different homologs requires the leucine-rich repeat receptor-like protein RCAP1, which associates with two central receptor-like kinases BRI1-associated receptor kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in planta. These findings suggest that the CAP-type apoplastic effectors act as an important player in plant-microbe interactions that can be perceived by plant membrane-localized receptor to activate plant resistance., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Plants can be infected by multiple pathogens concurrently in natural systems. However, pathogen-pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae-soybean-Fusarium combinations, more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen co-inoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1 (Fpp1), encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B 6 (VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of disease-resistant crop varieties and provide new strategies to control soybean root rot., (© 2023 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant-microbe interactions, but the underlying mechanisms remain elusive. Here, proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes., (© 2023. Springer Nature Limited.)

Plant pathogens secrete effector proteins to overcome host immunity and promote colonization. In oomycete plant pathogens, the expression of many effector genes is altered upon infection; however, the regulatory mechanisms are unclear. In this study, we identified a su(var)3-9, enhancer of zeste, and trithorax (SET) domain protein-encoding gene, PsKMT3, that was highly induced at early infection stages in Phytophthora sojae. Deletion of PsKMT3 led to asexual development and pathogenicity defects. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and western blot analyses demonstrated that histone H3K36 trimethylation (H3K36me3) was significantly reduced genome-wide in mutants. RNA-seq analysis identified 374 genes encoding secreted proteins that were differentially expressed in pskmt3 at the mycelium stage. The significantly altered genes encompassed the RxLR (Arg-x-Lys-Arg) effector gene family, including the essential effector genes Avh23, Avh181, Avh240, and Avh241. Transcriptome analysis at early infection stages showed misregulation of effector gene expression waves in pskmt3. H3K36me3 was directly and indirectly associated with RxLR effector gene activation. Our results reveal a role of a SET domain protein in regulating effector gene expression and modulating histone methylation in P. sojae., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor., Competing Interests: Conflict of interest statement. The authors have no conflicts of interest to declare., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nucleosome-free open chromatin often harbors transcription factor (TF)-binding sites that are associated with active cis -regulatory elements. However, analysis of open chromatin regions has rarely been applied to oomycete or fungal plant pathogens. In this study, we performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to identify open chromatin and cis -regulatory elements in Phytophthora sojae at the mycelial stage. We identified 10,389 peaks representing nucleosome-free regions (NFRs). The peaks were enriched in gene-promoter regions and associated with 40% of P. sojae genes; transcription levels were higher for genes with multiple peaks than genes with a single peak and were higher for genes with a single peak than genes without peak. Chromatin accessibility was positively correlated with gene transcription level. Through motif discovery based on NFR peaks in core promoter regions, 25 candidate cis -regulatory motifs with evidence of TF-binding footprints were identified. These motifs exhibited various preferences for location in the promoter region and associations with the transcription level of their target genes, which included some putative pathogenicity-related genes. As the first study revealing the landscape of open chromatin and the correlation between chromatin accessibility and gene transcription level in oomycetes, the results provide a technical reference and data resources for future studies on the regulatory mechanisms of gene transcription.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex plant-Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography-mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the β-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The 'zigzag' model formulates some of the fundamental principles underpinning the dynamic interactions between pathogen effectors and plant immunity. As key virulence factors, effectors often exhibit a pattern of rapid evolution, presumably as a result of the host-pathogen arms race. Here, we summarize the current knowledge of mechanisms that may accelerate effector evolution in the highly successful Phytophthora pathogens. Recent findings on epigenetic regulation of effector genes that allows evasion of host recognition and maintenance of cost/benefit balance, and a conserved structural unit in effector proteins that may promote the evolution of virulence activities are highlighted., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

Hosts and pathogens are engaged in a continuous evolutionary struggle for physiological dominance. A major site of this struggle is the apoplast. In Phytophthora sojae -soybean interactions, PsXEG1, a pathogen-secreted apoplastic endoglucanase, is a key focal point of this struggle, and the subject of two layers of host defense and pathogen counterdefense. Here, we show that N-glycosylation of PsXEG1 represents an additional layer of this coevolutionary struggle, protecting PsXEG1 against a host apoplastic aspartic protease, GmAP5, that specifically targets PsXEG1. This posttranslational modification also attenuated binding by the previously described host inhibitor, GmGIP1. N-glycosylation of PsXEG1 at N174 and N190 inhibited binding and degradation by GmAP5 and was essential for PsXEG1 's full virulence contribution, except in GmAP5-silenced soybeans. Silencing of GmAP5 reduced soybean resistance against WT P. sojae but not against PsXEG1 deletion strains of P. sojae. The crucial role of N-glycosylation within the three layers of defense and counterdefense centered on PsXEG1 highlight the critical importance of this conserved apoplastic effector and its posttranslational modification in Phytophthora -host coevolutionary conflict., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly., Competing Interests: The authors have declared that no competing interests exist.

Pathogen avirulence (Avr) effectors interplay with corresponding plant resistance (R) proteins and activate robust plant immune responses. Although the expression pattern of Avr genes has been tied to their functions for a long time, it is still not clear how Avr gene expression patterns impact plant-microbe interactions. Here, we selected PsAvr3b, which shows a typical effector gene expression pattern from a soybean root pathogen Phytophthora sojae. To modulate gene expression, we engineered PsAvr3b promoter sequences by in situ substitution with promoter sequences from Actin (constitutive expression), PsXEG1 (early expression), and PsNLP1 (later expression) using the CRISPR/Cas9. PsAvr3b driven by different promoters resulted in distinct expression levels across all the tested infection time points. Importantly, those mutants with low PsAvr3b expression successfully colonized soybean plants carrying the cognate R gene Rps3b. To dissect the difference in plant responses to the PsAvr3b expression level, we conducted RNA-sequencing of different infection samples at 24 h postinfection and found soybean immune genes, including a few previously unknown genes that are associated with resistance. Our study highlights that fine-tuning in Avr gene expression impacts the compatibility of plant disease and provides clues to improve crop resistance in disease control management., (© 2019 Institute of Botany, Chinese Academy of Sciences.)

The relentless adaptability of pathogen populations is a major obstacle to effective disease control measures. Increasing evidence suggests that gene transcriptional polymorphisms are a strategy deployed by pathogens to evade host immunity. However, the underlying mechanisms of transcriptional plasticity remain largely elusive. Here we found that the soybean root rot pathogen Phytophthora sojae evades the soybean Resistance gene Rps1b through transcriptional polymorphisms in the effector gene Avr1b that occur in the absence of any sequence variation. Elevated levels of histone H3 Lysine27 tri-methylation (H3K27me3) were observed at the Avr1b locus in a naturally occurring Avr1b-silenced strain but not in an Avr1b-expressing strain, suggesting a correlation between this epigenetic modification and silencing of Avr1b. To genetically test this hypothesis, we edited the gene, PsSu(z)12, encoding a core subunit of the H3K27me3 methyltransferase complex by using CRISPR/Cas9, and obtained three deletion mutants. H3K27me3 depletion within the Avr1b genomic region correlated with impaired Avr1b gene silencing in these mutants. Importantly, these mutants lost the ability to evade immune recognition by soybeans carrying Rps1b. These data support a model in which pathogen effector transcriptional polymorphisms are associated with changes in chromatin epigenetic marks, highlighting epigenetic variation as a mechanism of pathogen adaptive plasticity., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is a destructive disease afflicting soybean. The use of resistant cultivars is the most effective method to combat PRR. PRR resistance was assessed in 223 soybean cultivars from Huanghuaihai and Dongbei, major soybean-producing regions in east central and northeastern China. To evaluate levels of soybean resistance to P. sojae , we used eight representative P. sojae isolates and a modified etiolated hypocotyl-slit inoculation method. The cultivars Wandou21020, Xu9302-A, Kedou10, and Lidi055 showed resistance to all eight isolates; 14 cultivars showed intermediate resistance to all eight P. sojae isolates, and 53 cultivars were resistant to seven isolates. Thirty-three cultivars (15%) were susceptible only to the highly virulent PsJS2 isolate, which is consistent with the reactions of the Chapman differential line that carries Rps3a . The diverse reaction patterns seen in germplasm from different regions (provinces/cities) in this study reflect the variety of PRR-resistant soybean sources in China. Our research indicates that sources of P. sojae resistance are present in the major soybean production areas of China. This study provides useful information for soybean breeding programs.

Chitin is a structural and functional component of the fungal cell wall and also serves as a pathogen-associated molecular pattern (PAMP) that triggers the innate immune responses of host plants. However, no or very little chitin is found in the fungus-like oomycetes. In Phytophthora spp., the presence of chitin has not been demonstrated so far, although putative chitin synthase (CHS) genes, which encode the enzymes that synthesize chitin, are present in their genomes. Here, we revealed that chitin is present in the zoospores and released sporangia of Phytophthora, and this is most consistent with the transcriptional pattern of PcCHS in Phytophthora capsici and PsCHS1 in Phytophthora sojae. Disruption of the CHS genes indicated that PcCHS and PsCHS1, but not PsCHS2 (which exhibited very weak transcription), have similar functions involved in mycelial growth, sporangial production, zoospore release and the pathogenesis of P. capsici and P. sojae. We also suggest that chitin in the zoospores of P. capsici can act as a PAMP that is recognized by the chitin receptors AtLYK5 or AtCERK1 of Arabidopsis. These results provide new insights into the biological significance of chitin and CHSs in Phytophthora and help with the identification of potential targets for disease control., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Phytophthora pathogens secrete many effector proteins to manipulate host innate immunity. PsAvh238 is a Phytophthora sojae N-terminal Arg-X-Leu-Arg (RXLR) effector, which evolved to escape host recognition by mutating one nucleotide while retaining plant immunity-suppressing activity to enhance infection. However, the molecular basis of the PsAvh238 virulence function remains largely enigmatic. By using coimmunoprecipitation and liquid chromatography-tandem mass spectrometry analysis, we identified the 1-aminocyclopropane-1-carboxylate synthase (ACS) isoforms, the key enzymes in ethylene (ET) biosynthesis, as a host target of PsAvh238. We show that PsAvh238 interacts with soybean ACSs (GmACSs) in vivo and in vitro. By destabilizing Type2 GmACSs, PsAvh238 suppresses Type2 ACS-catalyzed ET biosynthesis and facilitates Phytophthora infection. Silencing of Type2 GmACSs, and inhibition of ET biosynthesis or signaling, increase soybean susceptibility to P. sojae infection, supporting a role for Type2 GmACSs and ET in plant immunity against P. sojae. Moreover, wild-type P. sojae but not the PsAvh238-disrupted mutants, inhibits ET induction and promotes P. sojae infection in soybean. Our results highlight the ET biosynthesis pathway as an essential part in plant immunity against P. sojae and a direct effector target., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six α helices and two WY motifs. The first two α helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Filamentous pathogens, such as fungi and oomycetes, secrete avirulence (AVR) effectors that trigger plant immune responses and provide striking examples of host adaptations. Avr effector genes display different types of allelic variations, including deletions, epigenetic silencing and sequence polymorphisms, to avoid detection. However, how effector sequence polymorphisms enable pathogens to dodge host immune surveillance remains largely unknown. PsAvr3c is a Phytophthora AVR gene that is recognized by soybean carrying Rps3c. PsAvr3c natural alleles display a rich diversity of single nucleotide polymorphisms in field isolates. We combined both site-directed mutagenesis and population sequence surveys to identify a serine substitution of glycine at position 174 in PsAvr3c that resulted in evasion of Rps3c-mediated soybean immunity. The S174G substitution did not affect the nuclear localization of PsAvr3c in planta, which is required to activate Rps3c, but it significantly impaired the binding affinity of PsAvr3c with a previously identified spliceosome-associated protein GmSKRPs. Silencing GmSKRPs specifically impaired PsAvr3c-triggered cell death in Rps3c soybean. This study uncovered a plant Phytophthora pathogen that adapted to a resistant plant through a key amino acid mutation and subsequently reduced the binding affinity with a plant immune regulator to evade host resistance., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Background: Filamentous plant pathogen genomes often display a bipartite architecture with gene-sparse, repeat-rich compartments serving as a cradle for adaptive evolution. The extent to which this two-speed genome architecture is associated with genome-wide DNA modifications is unknown., Results: We show that the oomycetes Phytophthora infestans and Phytophthora sojae possess functional adenine N6-methylation (6mA) methyltransferases that modulate patterns of 6mA marks across the genome. In contrast, 5-methylcytosine could not be detected in these species. Methylated DNA IP sequencing (MeDIP-seq) of each species reveals 6mA is depleted around the transcription start sites (TSSs) and is associated with lowly expressed genes, particularly transposable elements. Genes occupying the gene-sparse regions have higher levels of 6mA in both genomes, possibly implicating the methylome in adaptive evolution. All six putative adenine methyltransferases from P. infestans and P. sojae, except PsDAMT2, display robust enzymatic activities. Surprisingly, single knockouts in P. sojae significantly reduce in vivo 6mA levels, indicating that the three enzymes are not fully redundant. MeDIP-seq of the psdamt3 mutant reveals uneven 6mA methylation reduction across genes, suggesting that PsDAMT3 may have a preference for gene body methylation after the TSS. Furthermore, transposable elements such as DNA elements are more active in the psdamt3 mutant. A large number of genes, particularly those from the adaptive genomic compartment, are differentially expressed., Conclusions: Our findings provide evidence that 6mA modification is potentially an epigenetic mark in Phytophthora genomes, and complex patterns of 6mA methylation may be associated with adaptive evolution in these important plant pathogens.

Oomycete pathogens secrete host cell-entering effector proteins to manipulate host immunity during infection. We previously showed that PsAvh52, an early-induced RxLR effector secreted from the soybean root rot pathogen, Phytophthora sojae , could suppress plant immunity. Here, we found that PsAvh52 is required for full virulence on soybean and binds to a novel soybean transacetylase, GmTAP1, in vivo and in vitro. PsAvh52 could cause GmTAP1 to relocate into the nucleus where GmTAP1 could acetylate histones H2A and H3 during early infection, thereby promoting susceptibility to P. sojae . In the absence of PsAvh52, GmTAP1 remained confined to the cytoplasm and did not modify plant susceptibility. These results demonstrate that GmTAP1 is a susceptibility factor that is hijacked by PsAvh52 in order to promote epigenetic modifications that enhance the susceptibility of soybean to P. sojae infection., Competing Interests: HL, HW, MJ, JZ, BG, YW, YL, HC, LK, ZM, YW, WY, SD, BT, YW No competing interests declared, (© 2018, Li et al.)

The process of RNA splicing influences many physiological processes, including plant immunity. However, how plant parasites manipulate host RNA splicing process remains unknown. Here we demonstrate that PsAvr3c, an avirulence effector from oomycete plant pathogen Phytophthora sojae, physically binds to and stabilizes soybean serine/lysine/arginine-rich proteins GmSKRPs. The SKRPs are novel proteins that associate with a complex that contains plant spliceosome components, and are negative regulators of plant immunity. Analysis by RNA-seq data indicates that alternative splicing of pre-mRNAs from 401 soybean genes, including defense-related genes, is altered in GmSKRP1 and PsAvr3c overexpressing lines compared to control plants. Representative splicing events mediated by GmSKRP1 and PsAvr3c are tested by infection assays or by transient expression in soybean plants. Our results show that plant pathogen effectors can reprogram host pre-mRNA splicing to promote disease, and we propose that pathogens evolved such strategies to defeat host immune systems.

Immune response during pathogen infection requires extensive transcription reprogramming. A fundamental mechanism of transcriptional regulation is histone acetylation. However, how pathogens interfere with this process to promote disease remains largely unknown. Here we demonstrate that the cytoplasmic effector PsAvh23 produced by the soybean pathogen Phytophthora sojae acts as a modulator of histone acetyltransferase (HAT) in plants. PsAvh23 binds to the ADA2 subunit of the HAT complex SAGA and disrupts its assembly by interfering with the association of ADA2 with the catalytic subunit GCN5. As such, PsAvh23 suppresses H3K9 acetylation mediated by the ADA2/GCN5 module and increases plant susceptibility. Expression of PsAvh23 or silencing of GmADA2/GmGCN5 resulted in misregulation of defense-related genes, most likely due to decreased H3K9 acetylation levels at the corresponding loci. This study highlights an effective counter-defense mechanism by which a pathogen effector suppresses the activation of defense genes by interfering with the function of the HAT complex during infection., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Phytophthora pathogens secrete effectors to manipulate host innate immunity, thus facilitating infection. Among the RXLR effectors highly induced during Phytophthora sojae infection, Avh238 not only contributes to pathogen virulence but also triggers plant cell death. However, the detailed molecular basis of Avh238 functions remains largely unknown. We mapped the regions responsible for Avh238 functions in pathogen virulence and plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays. The correlation between cellular localization and Avh238 functions was also evaluated. We found that the 79 th residue (histidine or leucine) of Avh238 determined its cell death-inducing activity, and that the 53 amino acids in its C-terminal region are responsible for promoting Phytophthora infection. Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death, while Avh238-mediated suppression of INF1-triggered cell death requires cytoplasmic localization. Our results demonstrate that a representative example of an essential Phytophthora RXLR effector can evolve to escape recognition by the host by mutating one nucleotide site, and can also retain plant immunosuppressive activity to enhance pathogen virulence in planta., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae -soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae , however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems., (Copyright © 2017, American Association for the Advancement of Science.)

Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes.

Pathogen nutrient acquisition and metabolism are critical for successful infection and colonization. However, the nutrient requirements and metabolic pathways related to pathogenesis in oomycete pathogens are unknown. In this study, we bioinformatically identified Phytophthora sojae aspartate aminotransferases (AATs), which are key enzymes that coordinate carbon and nitrogen metabolism. We demonstrated that P. sojae encodes more AATs than the analysed fungi. Some of the AATs contained additional prephenate dehydratase and/or prephenate dehydrogenase domains in their N-termini, which are unique to oomycetes. Silencing of PsAAT3, an infection-inducible expression gene, reduced P. sojae pathogenicity on soybean plants and affected the growth under N-starving condition, suggesting that PsAAT3 is involved in pathogen pathogenicity and nitrogen utilisation during infection. Our results suggest that P. sojae and other oomycete pathogens may have distinct amino acid metabolism pathways and that PsAAT3 is important for its full pathogenicity., (Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

We identified a glycoside hydrolase family 12 (GH12) protein, XEG1, produced by the soybean pathogen Phytophthora sojae that exhibits xyloglucanase and β-glucanase activity. It acts as an important virulence factor during P. sojae infection but also acts as a pathogen-associated molecular pattern (PAMP) in soybean (Glycine max) and solanaceous species, where it can trigger defense responses including cell death. GH12 proteins occur widely across microbial taxa, and many of these GH12 proteins induce cell death in Nicotiana benthamiana. The PAMP activity of XEG1 is independent of its xyloglucanase activity. XEG1 can induce plant defense responses in a BAK1-dependent manner. The perception of XEG1 occurs independently of the perception of ethylene-inducing xylanase. XEG1 is strongly induced in P. sojae within 30 min of infection of soybean and then slowly declines. Both silencing and overexpression of XEG1 in P. sojae severely reduced virulence. Many P. sojae RXLR effectors could suppress defense responses induced by XEG1, including several that are expressed within 30 min of infection. Therefore, our data suggest that PsXEG1 contributes to P. sojae virulence, but soybean recognizes PsXEG1 to induce immune responses, which in turn can be suppressed by RXLR effectors. XEG1 thus represents an apoplastic effector that is recognized via the plant's PAMP recognition machinery., (© 2015 American Society of Plant Biologists. All rights reserved.)

Background: Basic leucine zipper (bZIP) transcription factors are present exclusively in eukaryotes and constitute one of the largest and most diverse transcription factor families. The proteins are responsible for central developmental and physiological processes in plants, animals, and fungi, including the pathogenicity of fungal plant pathogens. However, there is limited understanding of bZIPs in oomycetes, which are fungus-like organisms in the kingdom Stramenopila. Oomycetes include many destructive plant pathogens, including the well-studied species Phytophthora sojae, which causes soybean stem and root rot., Results: Candidate bZIPs encoded in the genomes of P. sojae and four other oomycetes, two diatoms, and two fungal species were predicted using bioinformatic methods. Comparative analysis revealed expanded numbers of bZIP candidates in oomycetes, especially the Phytophthora species, due to the expansion of several novel bZIP classes whose highly conserved asparagines in basic DNA-binding regions were substituted by other residues such as cysteine. The majority of these novel bZIP classes were mostly restricted to oomycetes. The large number of novel bZIPs appears to be the result of widespread gene duplications during oomycete evolution. The majority of P. sojae bZIP candidates, including both conventional and novel bZIP classes, were predicted to contain canonical protein secondary structures. Detection of gene transcripts using digital gene expression profiling and qRT-PCR suggested that most of the candidates were not pseudogenes. The major transcriptional shifts of bZIPs occurred during the zoosporangia/zoospore/cyst and host infection stages. Several infection-associated bZIP genes were identified that were positively regulated by H2O2 exposure., Conclusions: The identification of large classes of bZIP proteins in oomycetes with novel bZIP motif variants, that are conserved and developmentally regulated and thus presumably functional, extends our knowledge of this important family of eukaryotic transcription factors. It also lays the foundation for detailed studies of the roles of these proteins in development and infection in P. sojae and other oomycetes.

Soybean root and stem rot is caused by the oomycete pathogen Phytophthora sojae. The interaction between P. sojae and soybean fits the "gene-for-gene" hypothesis. Although more than 10 P. sojae avirulence (Avr) effectors have been genetically identified, nearly half of genetically defined avr genes have been cloned. In a previous bioinformatic and global transcriptional analysis, we identified a P. sojae RxLR effector, Avr1d, which was 125 amino acids in length. Mapping data demonstrated that Avr1d presence or absence in the genome was co-segregated with the Avr1d avirulence phenotype in F2 populations. Transient expression of the Avr1d gene using co-bombardment in soybean isogenic lines revealed that this gene triggered a hypersensitive response (HR) in the presence of Rps1d. Sequencing of Avr1d genes in different P. sojae strains revealed two Avr1d alleles. Although polymorphic, the two Avr1d alleles could trigger Rps1d-mediated HR. P. sojae strains carrying either of the alleles were avirulent on Rps1d soybean lines. Avr1d was upregulated during the germinating cyst and early infection stages. Furthermore, transient expression of Avr1d in Nicotiana benthamiana suppressed BAX-induced cell death and enhanced P. capsici infection. Avr1d also suppressed effector-triggered immunity induction by associating with Avr1b and Rps1b, suggestive of a role in suppressing plant immunity.

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae, Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

The effector gene Avr1b-1 of Phytophthora sojae determines the efficacy of the resistance gene Rps1b in soybean. The sequences of the Avr1b-1 locus in 34 Chinese isolates of P. sojae were obtained and analysed by polymerase chain reaction (PCR) and inverse PCR. Four different alleles and a complete deletion mutation of the Avr1b-1 gene were identified. Molecular analysis of the deletion breakpoints in the Avr1b-1 locus revealed that an 8-kb DNA sequence containing Avr1b-1 was deleted and a 12.7-kb DNA sequence was inserted at the same locus. A truncated transposase gene was found and five transposable elements were predicted in the inserted sequence, suggesting that the deletion of Avr1b-1 might be attributed to transposon movement. The transcription of Avr1b-1 was analysed in virulent isolates containing four alleles of Avr1b-1 by real-time reverse transcription-PCR. In all virulent isolates, only those isolates containing the second allele transcripted Avr1b-1., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at ten different developmental and infection stages based on a 3'-tag digital gene-expression protocol. More than 90 million clean sequence tags were generated and compared with the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A comparison of the whole-library genes' expression patterns suggested four groups: i) mycelia and zoosporangia, ii) zoospores and cysts, iii) germinating cysts, and iv) five infection site libraries (IF1.5 to IF24h). The libraries from the different groups showed major transitional shifts in gene expression. From the ten libraries, 722 gene expression?pattern clusters were obtained and the top 16 clusters, containing more than half of the genes, comprised enriched genes with different functions including protein localization, triphosphate metabolism, signaling process, and noncoding RNA metabolism. An evaluation of the average expression level of 30 pathogenesis-related gene families revealed that most were infection induced but with diverse expression patterns and levels. A web-based server named the Phytophthora Transcriptional Database has been established.

In many eukaryotic organisms, Cdc14 phosphatase regulates multiple biological events during anaphase and is essential for mitosis. It has been shown that Cdc14 is required for sporulation in the potato blight pathogen Phytophthora infestans; however, the role that the Cdc14 homolog (PsCdc14) plays in the soil-borne soybean root rot pathogen P. sojae remains ambiguous. PsCdc14 is highly expressed in sporulation, zoospore, and cyst life stages, but not in vegetative mycelia and infection stages, suggesting that it contributes to asexual reproduction and thus the spread of the disease. Double-stranded RNA (dsRNA) mediates gene silencing, a post-transcriptional and highly conserved process in eukaryotes, involving specific gene silencing through degradation of target mRNA. We combined in vitro dsRNA synthesis and a polyethylene glycol-mediated transformation system to construct a dsRNA-mediated transient gene silencing system; and then performed a functional analysis of PsCdc14 in P. sojae. PsCdc14 mRNA was dramatically reduced in transformants after protoplasts were exposed in in vitro synthesized PsCdc14 dsRNA, resulting in low sporangial production and abnormal development in P. sojae silencing lines. Furthermore, dsRNA-mediated transient gene silencing could enable elucidation of P. sojae rapid gene function, facilitating our understanding of the development and pathogenicity mechanisms of this oomycete fungus.

Plants have evolved pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to protect themselves from infection by diverse pathogens. Avirulence (Avr) effectors that trigger plant ETI as a result of recognition by plant resistance (R) gene products have been identified in many plant pathogenic oomycetes and fungi. However, the virulence functions of oomycete and fungal Avr effectors remain largely unknown. Here, we combined bioinformatics and genetics to identify Avr3b, a new Avr gene from Phytophthora sojae, an oomycete pathogen that causes soybean root rot. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Some isolates of P. sojae evade perception by the soybean R gene Rps3b through sequence mutation in Avr3b and lowered transcript accumulation. Transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P. parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Biochemical assays confirmed that Avr3b is an ADP-ribose/NADH pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif of Avr3b abolished enzyme activity. Mutation of key residues in Nudix motif significantly impaired Avr3b virulence function but not the avirulence activity. Some Nudix hydrolases act as negative regulators of plant immunity, and thus Avr3b might be delivered into host cells as a Nudix hydrolase to impair host immunity. Avr3b homologues are present in several sequenced Phytophthora genomes, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity.

The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The oomycetes, a distinct phylogenetic lineage of fungus-like microorganisms, are heterokonts (stramenopiles) belonging to the supergroup Chromalveolata. Although the complete genomic sequences of a number of oomycetes have been reported, little information regarding the introns therein is available. Here, we investigated the introns of Phytophthora sojae, a pathogen that causes soybean root and stem rot, by a comparative analysis of genomic sequences and expressed sequence tags. A total of 4013 introns were identified, of which 96.6% contained canonical splice sites. The P. sojae genome possessed features distinct from other organisms at 5' splice sites, polypyrimidine tracts, branch sites, and 3' splice sites. Diverse repeating sequences, ranging from 2 to 10 nucleotides in length, were found at more than half of the intron-exon boundaries. Furthermore, 122 genes underwent alternative splicing. These data indicate that P. sojae has unique splicing mechanisms, and recognition of those mechanisms may lead to more accurate predictions of the location of introns in P. sojae and even other oomycete species.

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F(2) progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.

Mitogen-activated protein kinase (MAPK) pathways are universal and evolutionarily conserved signal transduction modules in all eukaryotic cells. In this study, PsSAK1, which encodes a stress-activated MAPK of Phytophthora sojae, was identified. PsSAK1 is highly conserved in oomycetes, and it represents a novel group of MAPK due to its pleckstrin homology domain. Reverse-transcription polymerase chain reaction analysis showed that PsSAK1 expression was upregulated in zoospores and cysts and during early infection. In addition, its expression was induced by osmotic and oxidative stress mediated by NaCl and H(2)O(2), respectively. To elucidate the function, the expression of PsSAK1 was silenced using stable transformation of P. sojae. The silencing of PsSAK1 did not impair hyphal growth, sporulation, or oospore production but severely hindered zoospore development, in that the silenced strains showed quicker encystment and a lower germination ratio than the wild type. PsSAK1-silenced mutants produced much longer germ tubes and could not colonize either wounded or unwounded soybean leaves. Our results indicate that PsSAK1 is an important regulator of zoospore development and pathogenicity in P. sojae.

Root and stem rot disease of soybean is caused by the oomycete Phytophthora sojae. The avirulence (Avr) genes of P. sojae control race-cultivar compatibility. In this study, we identify the P. sojae Avr3c gene and show that it encodes a predicted RXLR effector protein of 220 amino acids. Sequence and transcriptional data were compared for predicted RXLR effectors occurring in the vicinity of Avr4/6, as genetic linkage of Avr3c and Avr4/6 was previously suggested. Mapping of DNA markers in a F(2) population was performed to determine whether selected RXLR effector genes co-segregate with the Avr3c phenotype. The results pointed to one RXLR candidate gene as likely to encode Avr3c. This was verified by testing selected genes by a co-bombardment assay on soybean plants with Rps3c, thus demonstrating functionality and confirming the identity of Avr3c. The Avr3c gene together with eight other predicted genes are part of a repetitive segment of 33.7 kb. Three near-identical copies of this segment occur in a tandem array. In P. sojae strain P6497, two identical copies of Avr3c occur within the repeated segments whereas the third copy of this RXLR effector has diverged in sequence. The Avr3c gene is expressed during the early stages of infection in all P. sojae strains examined. Virulent alleles of Avr3c that differ in amino acid sequence were identified in other strains of P. sojae. Gain of virulence was acquired through mutation and subsequent sequence exchanges between the two copies of Avr3c. The results illustrate the importance of segmental duplications and RXLR effector evolution in the control of race-cultivar compatibility in the P. sojae and soybean interaction.

The importance of segmental duplications and copy number variants as a source of genetic and phenotypic variation is gaining greater appreciation, in a variety of organisms. Now, we have identified the Phytophthora sojae avirulence genes Avr1a and Avr3a and demonstrate how each of these Avr genes display copy number variation in different strains of P. sojae. The Avr1a locus is a tandem array of four near-identical copies of a 5.2 kb DNA segment. Two copies encoding Avr1a are deleted in some P. sojae strains, causing changes in virulence. In other P. sojae strains, differences in transcription of Avr1a result in gain of virulence. For Avr3a, there are four copies or one copy of this gene, depending on the P. sojae strain. In P. sojae strains with multiple copies of Avr3a, this gene occurs within a 10.8 kb segmental duplication that includes four other genes. Transcriptional differences of the Avr3a gene among P. sojae strains cause changes in virulence. To determine the extent of duplication within the superfamily of secreted proteins that includes Avr1a and Avr3a, predicted RXLR effector genes from the P. sojae and the P. ramorum genomes were compared by counting trace file matches from whole genome shotgun sequences. The results indicate that multiple, near-identical copies of RXLR effector genes are prevalent in oomycete genomes. We propose that multiple copies of particular RXLR effectors may contribute to pathogen fitness. However, recognition of these effectors by plant immune systems results in selection for pathogen strains with deleted or transcriptionally silenced gene copies.

Summary: The regulatory roles of RNA splicing in plant immunity are emerging but still largely obscure. We reported previously that Phytophthora pathogen effector Avr3c targets a soybean protein SKRP (serine/lysine/arginine‐rich protein) to impair soybean basal immunity by regulating host pre‐mRNA alternative splicing, while the biochemical nature of SKRP remains unknown.Here, by using Arabidopsis as a model, we studied the mechanism of SKRP in regulating pre‐mRNA splicing and plant immunity.AtSKRP confers impaired plant immunity against Phytophthora capsici and associates with spliceosome component PRP8 and splicing factor SR45, which positively and negatively regulate plant immunity, respectively. Enhanced crosslinking and immunoprecipitation followed by high‐throughput sequencing (eCLIP‐seq) showed AtSKRP is a novel RNA‐binding protein that targets exon 3′ end of unspliced RNA. Such position‐specific binding of SKRP is associated with its activity in suppressing intron retention, including at positive immune regulatory genes UBP25 and RAR1. In addition, we found AtSKRP self‐interact and forms oligomer, and these properties are associated with its function in plant immunity.Overall, our findings reveal that the immune repressor SKRP is a spliceosome‐associated protein that targets exon 3′ end to regulate pre‐mRNA splicing in Arabidopsis. [ABSTRACT FROM AUTHOR]

Summary: The plant apoplast is a harsh environment in which hydrolytic enzymes, especially proteases, accumulate during pathogen infection. However, the defense functions of most apoplastic proteases remain largely elusive.We show here that a newly identified small cysteine‐rich secreted protein PC2 from the potato late blight pathogen Phytophthora infestans induces immunity in Solanum plants only after cleavage by plant apoplastic subtilisin‐like proteases, such as tomato P69B.A minimal 61 amino acid core peptide carrying two key cysteines, conserved widely in most oomycete species, is sufficient for PC2‐induced cell death. Furthermore, we showed that Kazal‐like protease inhibitors, such as EPI1, produced by P. infestans prevent PC2 cleavage and dampen PC2 elicited host immunity.This study reveals that cleavage of pathogen proteins to release immunogenic peptides is an important function of plant apoplastic proteases. [ABSTRACT FROM AUTHOR]