Profiling of Phakopsora pachyrhizi transcriptome revealed co-expressed virulence effectors as prospective RNA interference targets for soybean rust management.

Soybean rust (SBR), caused by an obligate biotrophic pathogen Phakopsora pachyrhizi, is a devastating disease of soybean worldwide. However, the mechanisms underlying plant invasion by P. pachyrhizi are poorly understood, which hinders the development of effective control strategies for SBR. Here we performed detailed histological characterization on the infection cycle of P. pachyrhizi in soybean and conducted a high-resolution transcriptional dissection of P. pachyrhizi during infection. This revealed P. pachyrhizi infection leads to significant changes in gene expression with 10 co-expressed gene modules, representing dramatic transcriptional shifts in metabolism and signal transduction during different stages throughout the infection cycle. Numerous genes encoding secreted protein are biphasic expressed, and are capable of inhibiting programmed cell death triggered by microbial effectors. Notably, three co-expressed P. pachyrhizi apoplastic effectors (PpAE1, PpAE2, and PpAE3) were found to suppress plant immune responses and were essential for P. pachyrhizi infection. Double-stranded RNA coupled with nanomaterials significantly inhibited SBR infection by targeting PpAE1, PpAE2, and PpAE3, and provided long-lasting protection to soybean against P. pachyrhizi. Together, this study revealed prominent changes in gene expression associated with SBR and identified P. pachyrhizi virulence effectors as promising targets of RNA interference-based soybean protection strategy against SBR.

Plant immunity suppressor SKRP encodes a novel RNA-binding protein that targets exon 3' end of unspliced RNA.

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.

N-glycosylation shields Phytophthora sojae apoplastic effector PsXEG1 from a specific host aspartic protease.

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.

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages.

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.

Effector gene silencing mediated by histone methylation underpins host adaptation in an oomycete plant pathogen.

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.

Transcription factor MoMsn2 targets the putative 3-methylglutaconyl-CoA hydratase-encoding gene MoAUH1 to govern infectious growth via mitochondrial fusion/fission balance in Magnaporthe oryzae.

Mitochondrial quality and quantity are essential for a cell to maintain normal cellular functions. Our previous study revealed that the transcription factor MoMsn2 plays important roles in the development and virulence of Magnaporthe oryzae. However, to date, no study has reported its underlying regulatory mechanism in phytopathogens. Here, we explored the downstream target genes of MoMsn2 using a chromatin immunoprecipitation sequencing (ChIP-Seq) approach. In total, 332 target genes and five putative MoMsn2-binding sites were identified. The 332 genes exhibited a diverse array of functions and the highly represented were genes involved in metabolic and catalytic processes. Based on the ChIP-Seq data, we found that MoMsn2 plays a role in maintaining mitochondrial morphology, likely by targeting a number of mitochondria-related genes. Further investigation revealed that MoMsn2 targets the putative 3-methylglutaconyl-CoA hydratase-encoding gene (MoAUH1) to control mitochondrial morphology and mitophagy, which are critical for the infectious growth of the pathogen. Meanwhile, the deletion of MoAUH1 resulted in phenotypes similar to the ΔMomsn2 mutant in mitochondrial morphology, mitophagy and virulence. Overall, our results provide evidence for the regulatory mechanisms of MoMsn2, which targets MoAUH1 to modulate its transcript levels, thereby disturbing the mitochondrial fusion/fission balance. This ultimately affects the development and virulence of M. oryzae.

Cleavage of a pathogen apoplastic protein by plant subtilases activates host immunity.

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.

Editing of an effector gene promoter sequence impacts plant-Phytophthora interaction.

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.

The SET domain protein PsKMT3 regulates histone H3K36 trimethylation and modulates effector gene expression in the soybean pathogen Phytophthora sojae.

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.

Divergent sequences of tetraspanins enable plants to specifically recognize microbe-derived extracellular vesicles.

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.

Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions.

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.