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Many species in the fungal Diaporthe (anamorph Phomopsis) genus have become a group of the most important pathogens that cause seed decay, stem and pot blight, and stem canker in soybean production worldwide, resulting in significant yield loss. Due to increased disease prevalence but a lack of research, we performed an extensive field survey to isolate and identify the Diaporthe species associated with soybean stem blight in six provinces of China between 2019 and 2020. A total of 92 Diaporthe isolates were identified based on morphological and multilocus phylogenetic analysis and classified into six species: D. longicolla, D. unshiuensis, D. sojae, D. caulivora, D. tectonigena, and an unknown Diaporthe sp. The most frequently identified species was D. longicolla with 57 isolates. High genetic diversity was observed for the D. longicolla isolates, and haplotype network analysis revealed a mixed structure among the population in the six provinces. In comparative pathogenicity assays, different virulence levels were observed among the 92 Diaporthe isolates. The results of this study provide new insights into the Diaporthe spp. associated with soybean stem blight in China and can help in the development of effective management strategies.

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

While conventional chemical fungicides directly eliminate pathogens, plant immunity inducers activate or prime plant immunity. In recent years, considerable progress has been made in understanding the mechanisms of immune regulation in plants. The development and application of plant immunity inducers based on the principles of plant immunity represent a new field in plant protection research. In this review, we describe the mechanisms of plant immunity inducers in terms of plant immune system activation, summarize the various classes of reported plant immunity inducers (proteins, oligosaccharides, chemicals, and lipids), and review methods for the identification or synthesis of plant immunity inducers. The current situation, new strategies, and future prospects in the development and application of plant immunity inducers are also discussed.

Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense’ are closely related species and important pathogens of soybean that cause root rot. However, the sequences of commonly used molecular markers, such as rDNA internal transcribed spacer 2 and cytochrome oxidase 1 gene, are similar among these species, making it difficult to design species-specific primers for loop-mediated isothermal amplification (LAMP) assays. The genome sequences of these species are also currently unavailable. Based on a comparative genomic analysis and de novo RNA-sequencing transcript assemblies, we identified and cloned the sequences of the M90 gene, a conserved but highly polymorphic single-copy gene encoding a Puf family RNA-binding protein among oomycetes. After primer design and screening, three LAMP assays were developed that specifically amplified the targeted DNA sequences in P. terrestris and P. spinosum at 62°C for 70 min and in ‘Ca. Pythium huanghuaiense’ at 62°C for 60 min. After adding SYBR Green I, a positive yellow-green color (under natural light) or intense green fluorescence (under ultraviolet light) was observed by the naked eye only in the presence of the target species. The minimum concentration of target DNA detected in all three LAMP assays was 100 pg·μl−1. The assays also successfully detected the target Pythium spp. with high accuracy and sensitivity from inoculated soybean seedlings and soils collected from soybean fields. This study provides a method for identification and cloning of candidate detection targets without a reference genome sequence and identified M90 as a novel specific target for molecular detection of three Pythium species causing soybean root rot.

Plants deploy various immune receptors to recognize pathogen-derived extracellular signals and subsequently activate the downstream defense response. Recently, increasing evidence indicates that the endoplasmic reticulum (ER) plays a part in the plant defense response, known as ER stress-mediated immunity (ERSI), that halts pathogen infection. However, the mechanism for the ER stress response to signals of pathogen infection remains unclear. Here, we characterized the ER stress response regulator NAC089, which was previously reported to positively regulate programed cell death (PCD), functioning as an ERSI regulator. NAC089 translocated from the ER to the nucleus via the Golgi in response to Phytophthora capsici culture filtrate (CF), which is a mixture of pathogen-associated molecular patterns (PAMPs). Plasma membrane localized co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) was required for the CF-mediated translocation of NAC089. The nuclear localization of NAC089, determined by the NAC domain, was essential for immune activation and PCD. Furthermore, NAC089 positively contributed to host resistance against the oomycete pathogen P. capsici and the bacteria pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. We also proved that NAC089-mediated immunity is conserved in Nicotiana benthamiana. Together, we found that PAMP signaling induces the activation of ER stress in plants, and that NAC089 is required for ERSI and plant resistance against pathogens.

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.

Significance The apoplastic space is the initial battlefield in plant–microbe interactions. However, the molecular mechanisms underlying how apoplastic immunity controls pathogen invasion is still largely unknown. Here, we show that soybean secretes an apoplastic aspartic protease, GmAP5, that binds to and degrades PsXEG1 to block its contribution to virulence. Phytophthora sojae, however, employs N-glycosylation as a shield to protect PsXEG1 from degradation by GmAP5. N-glycosylation of PsXEG1 also attenuates the binding by the inhibitor GmGIP1. Our result uncovers an additional layer of defense and counterdefense centered on PsXEG1, highlighting an example in which N-glycosylation of a pathogen virulence factor tips the balance of an arms race in host–pathogen conflicts., 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.

Roots hold complex microbial communities at the soil–root interface, which can affect plant nutrition, growth, and health. Although the composition of plant microbiomes has been extensively described for various plant species and environments, little is known about the effect of wheat straw return (WSR) on the soybean root microbiota. We used Illumina-based 16S rRNA and ITS amplicon sequencing to track changes in bacterial and fungal microbiota in bulk soil and soybean rhizosphere, rhizoplane, s1and endosphere during the third and fourth years after implementing WSR in a wheat–soybean rotation system. The results revealed that WSR had a greater impact on fungal communities than bacterial communities, particularly in bulk soil, rhizosphere, and rhizoplane. WSR enriched the relative abundance of cellulose-degrading fungi (e.g., Acremonium, Trichoderma, and Myrmecridium, among which Trichoderma also had antimicrobial activity), saprotroph (e.g., Exophiala), and nitrogen cycling bacteria (e.g., Chryseolinea). Furthermore, WSR depleted the relative abundance of pathogenic fungi (e.g., Fusarium and Alternaria). These data revealed for the first time that WSR had diverse effects on soybean root-associated microbial community composition, not only in soil but also in the rhizosphere, rhizoplane, and endosphere.

Soybean is one of the most valuable legume crops in the world with high nutritional value. Recently, the outbreak of soybean stay-green syndrome has swept the soybean production in the Huang-Huai-Hai region of China, resulting in huge yield losses, which has become an epidemic and prominent problem in soybean production. However, the cause of the stay-green syndrome remains obscure. Here, we report a novel intergeneric recombinant geminivirus which causes soybean stay-green symptoms. Viral small RNA-based screening identified a new recombinant geminvirus from field soybean stay-green samples. The complete genome sequence of the virus contains 2762 nucleotide (nt) and appears to be an intergeneric recombinant virus in which protein coding for coat protein (V1) is similar to member of genus Mastrevirus, whereas proteins coding for V2, C2, C3 are most similar to those of viruses in the Maldovirus genus, and C1 and C4 are most similar to virus in genus Begomovirus. Inoculation of the infectious clone of the recombinant geminivirus through Agrobacterium rhizogenes causes typical soybean stay-green syndrome which resembles field symptoms including delayed leaf senescence, flat pods and abnormal seeds. The recombinant geminivirus can be detected in seed coat but not in cotyledon and embryo, thus failing to be transmitted by seeds. Moreover, the genome variation and epidemiological dynamic analysis were also carried out to help the continuous epidemiological surveillance of this emerging geminivirus. Collectively, this new geminivirus is tentatively named soybean stay-green associated virus (SoSGV). Our determination of the causal agent of soybean stay-green disease will bolster efforts to develop effective management strategies to control this prevalent disease in the field.

Soybean root diseases are associated with numerous fungal and oomycete pathogens; however, the community dynamics and interactions of these pathogens are largely unknown. We performed 13 loop-mediated isothermal amplification (LAMP) assays that targeted specific soybean root pathogens, and traditional isolation assays. A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages (30, 60, and 90 days after planting) in 2016. In LAMP results, we found that pathogen communities differed slightly among locations, but changed dramatically between soybean growth stages. Phytophthora sojae, Rhizoctonia solani, and Fusarium oxysporum were most frequently detected at the early stage, whereas Phomopsis longicolla, Fusarium equiseti, and Fusarium virguliforme were most common in the later stages. Most samples (86%) contained two to six pathogen species. Interestingly, the less detectable species tended to exist in the samples containing more detected species, and some pathogens preferentially co-occurred in diseased tissue, including P. sojae–R. solani–F. oxysporum and F. virguliforme–Calonectria ilicicola, implying potential interactions during infection. The LAMP detection results were confirmed by traditional isolation methods. The isolated strains exhibited different virulence to soybean, further implying a beneficial interaction among some pathogens.

The migratory plant‐parasitic nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which causes serious damage to pine forests in China. Plant immunity plays an important role in plant resistance to multiple pathogens. Activation of the plant immune system is generally determined by immune receptors, including plant pattern recognition receptors, which mediate pattern recognition. However, little is known about molecular pattern recognition in the interaction between pines and B. xylophilus. Based on the B. xylophilus transcriptome at the early stages of infection and Agrobacterium tumefaciens‐mediated transient expression and infiltration of recombinant proteins produced by Pichia pastoris in many plant species, a novel molecular pattern (BxCDP1) was characterized in B. xylophilus. We found that BxCDP1 was highly up‐regulated at the early infection stages of B. xylophilus, and was similar to a protein in Pararhizobium haloflavum. BxCDP1 triggered cell death in Nicotiana benthamiana when secreted into the apoplast, and this effect was dependent on brassinosteroid‐insensitive 1‐associated kinase 1, but independent of suppressor of BIR1‐1. BxCDP1 also exhibited cell death‐inducing activity in pine, Arabidopsis, tomato, pepper, and lettuce. BxCDP1 triggered reactive oxygen species production and the expression of PAMP‐triggered immunity marker genes (NbAcre31, NbPTI5, and NbCyp71D20) in N. benthamiana. It also induced the expression of pathogenesis‐related genes (PtPR‐3, PtPR‐4, and PtPR‐5) in Pinus thunbergii. These results suggest that as a new B. xylophilus molecular pattern, BxCDP1 can not only be recognized by many plant species, but also triggers innate immunity in N. benthamiana and defence responses of P. thunbergii., BxCDP1 can not only trigger innate immunity of the nonhost plant Nicotiana benthamiana, which depends on co‐receptors NbBAK1, but also induces defence responses of the host Pinus thunbergii.

Soybean contains a group of 64 L-type lectin receptor-like kinases. Three LecRKs were involved in the interactions with Phytophthora sojae and Bradyrhizobium diazoefficiens. L-type lectin receptor-like kinases (LecRKs) comprise an important class of membrane-localized receptor-like kinases that are involved in plant adaptation. In this study, we performed an inventory analysis of LecRKs in Glycine max (soybean). In total, 64 GmLecRKs containing the canonical LecRK feature were identified. Phylogenetic analysis revealed that 48 GmLecRKs have close orthologs in Arabidopsis or Solanum lycopersicum, while 16 are likely present only in the leguminous plant species. Transcriptome analyses revealed that expressions of multiple GmLecRK genes are either induced or suppressed during infection by the soybean root rot pathogen Phytophthora sojae. In addition, overexpression of the three LecRKs (Glyma.17G085000, Glyma.05G041300 or Glyma.17G224600) in the soybean hairy roots enhanced resistance to P. sojae. Upon inoculation with Bradyrhizobium diazoefficiens, overexpression of Glyma.17G085000 in the soybean hairy roots does not significantly influence the nodulation, while overexpression of Glyma.05G041300 or Glyma.17G224600 slightly reduced the number and dry weight of nodules. This study highlights the importance of LecRKs in regulating plant–microbe interactions and provides new knowledge on the deployment of LecRKs to increase resistance in soybean.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway., Author summary P. sojae secretes RxLR effectors to modulate host plant immunity. PsAvh181, an atypical RxLR effector of P. sojae, contributes to the full virulence of P. sojae. However, the mechanism by which PsAvh181 modulates plant immunity remains unknown. We found that PsAvh181 localizes to the plant plasma membrane to inhibit the secretion of apoplastic defense-related proteins such as GmGIP1, P69B and PR1. We screened target proteins of PsAvh181 by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry analyses. It was found that PsAvh181 binds to a soybean soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (GmSNAP-1), which is an important component of the SNAP receptor (SNARE) complex and plays a significant role in vesicle trafficking. Further investigation showed that PsAvh181 interferes the interaction between GmSNAP-1 and GmNSF, thus blocking the secretion of defense-related proteins and thereby modulating plant immunity. Our results reveal how a P. sojae effector suppresses plant immunity by inhibiting the secretion of apoplastic defense-related proteins.

Candidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen that is associated with the devastating citrus Huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful and dual-transcriptome analyses aiming to profile gene expression in both Las and its host(s) have a low coverage of the Las genome due to the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, the lack of a Las transcriptome remains as a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited the highest expression levels in citrus include ion transporters, ferritin, outer membrane porins, and genes involved in phage-related functions, pilus formation, cell wall modification, and stress responses. One hundred and six genes were found to be differentially expressed in citrus vs psyllids. Genes related to transcription/translation and resilience to host defense response were upregulated in citrus; whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. We also determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Plant-pathogenic oomycetes include numerous species that are ongoing threats to agriculture and natural ecosystems. Understanding the molecular dialogs between oomycetes and plants is instrumental for sustaining effective disease control. Plants respond to oomycete infection by multiple defense actions including strengthening of physical barriers, production of antimicrobial molecules, and programmed cell death. These responses are tightly controlled and integrated via a three-layered immune system consisting of a multiplex recognition layer, a resilient signal-integration layer, and a diverse defense-action layer. Adapted oomycete pathogens utilize apoplastic and intracellular effector arsenals to counter plant immunity mechanisms within each layer, including by evasion or suppression of recognition, interference with numerous signaling components, and neutralization or suppression of defense actions. A coevolutionary arms race continually drives the emergence of new mechanisms of plant defense and oomycete counterdefense.

Phytophthora sojae is a destructive pathogen of soybean that is widely distributed in the world. The interaction between P. sojae and soybean follows the gene-for-gene model. The use of resistant soybean cultivars is the primary and most effective method to combat the disease. However, variation in the Avr genes of the pathogen enables it to evade host defenses. We collected 81 isolates from four major soybean-production areas in China to analyze the polymorphism of Avr genes in P. sojae field population. The virulence of these isolates towards 14 differential soybean lines indicated complex pathotypes in P. sojae field population in China. In this study we found that Rps1c, which is cognate with Avr1c, could be deployed in Heilongjiang, Shandong, and Jiangsu Provinces but not in Anhui Province. To determine the mechanism by which Avr1c escapes recognition by Rps1c, we analyzed the polymorphism of Avr1c gene in 50 isolates of a field population of P. sojae and found multiple novel genotypes related to virulence and avirulence. By performing infection assays and gene co-bombardment, we showed that the K105 amino-acid residue was under strong positive selection and was a determinant of the avirulence of Avr1c. Structural analysis showed that K105 was exposed on the surface of the protein, suggesting it to be a critical site for interacting with Rps genes or their associated proteins.

The oomycete pathogen causing root-rot and damping-off diseases in soybean (Glycine max) fields in the Huang-Huai region of China was identified as Pythium spinosum. To detect P. spinosum for disease diagnosis and control, we developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA internal transcribed spacer 2 (ITS2) sequence of P. spinosum. The LAMP assay can efficiently amplify the target gene within 60 min at 63 °C. In specificity tests using 31 Pythium spp., 12 Phytophthora spp., 6 Phytopythium spp., and 9 other fungi strains, no cross-reactions were observed in the LAMP assay. The detection limit was 100 pg·μL−1 of genomic DNA per reaction. In cases of suspected disease, P. spinosum could be detected directly, using the LAMP assay, from soybean tissues and soil collected from fields in soybean production areas in the Huang-Huai region. This study provides a rapid method for diagnosing soybean root-rot and damping-off diseases caused by P. spinosum.

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.

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.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1–associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

Strigolactones inhibit bud growth by negatively regulating the auxin transport without changing the auxin biosynthesis and suppressing the expression of A-ARR in buds. Strigolactones (SLs) are important phytohormones associated with regulation of shoot branching in rice. Rice shoot branching is persuasively mediated by plant hormones like auxin, cytokinins (CKs) and SLs. The interactions among these hormones were diversely investigated by many researchers but remained a subject of debate. In the present study, the removal of panicle and application of subsequent synthetic SLs were used to regulate rice bud growth on node 2 (the second node from panicle) at full heading stage. The bud growth was significantly induced after panicle removal but GR24 (synthetic SLs) application inhibited it, along with variations in endogenous hormone contents in bud. RNA samples from buds were subjected to RNA sequencing through Illumina HiSeq 2000 (RNA-seq). Comparison of transcript expression levels among three treatments, viz. (1) intact (Co), (2) removed panicle (RP) and (3) RP combined with synthetic SL GR24 (GR) revealed the involvement of numerous genes associated with hormone signal transduction. GR24 supply minimized the RP-induced enhancement of auxin early response genes, independent of ARF. CK signal transduction was also induced by RP, but type-A ARR were the only genes responding to GR without any other CK signal associated genes. Additionally, RP and GR can also modulate auxin transport and CK degradation by regulating the genes' expression involved in the biosynthesis of flavonoid, phenylpropanoid and benzoxazinoid. Contemplating the results obtained so far, it is possible to open new vistas of research to reveal the interactions among SLs, auxin and CK in controlling the shoot branching of rice.

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.

Significance Ubiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijack the host ubiquitination system. However, the molecular mechanisms by which effectors modulate the host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box-type E3 ligase GmPUB13, which proved to be a susceptibility factor. The crystal structure of Avr1d complexed with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for E2 ubiquitin conjugating enzyme and competes with E2 for binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study reveals a structural basis for modulation of host targets by Phytophthora effectors., Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

Due to their sessile nature, plants must respond to various environmental assaults in a coordinated manner. The endoplasmic reticulum is a central hub for plant responses to various stresses. We previously showed that Phytophthora utilizes effector PsAvh262-mediated binding immunoglobulin protein (BiP) accumulation for suppressing endoplasmic reticulum stress-triggered cell death. As a BiP binding partner, Bcl-2-associated athanogene 7 (BAG7) plays a crucial role in the maintenance of the unfolded protein response, but little is known about its role in plant immunity. In this work, we reveal a double-faced role of BAG7 in Arabidopsis–Phytophthora interaction in which it regulates endoplasmic reticulum stress-mediated immunity oppositely in different cellular compartments. In detail, it acts as a susceptibility factor in the endoplasmic reticulum, but plays a resistance role in the nucleus against Phytophthora. Phytophthora infection triggers the endoplasmic reticulum-to-nucleus translocation of BAG7, the same as abiotic heat stress; however, this process can be prevented by PsAvh262-mediated BiP accumulation. Moreover, the immunoglobulin/albumin-binding domain in PsAvh262 is essential for both pathogen virulence and BiP accumulation. Taken together, our study uncovers a double-faced role of BAG7; Phytophthora advances its colonization in planta by utilizing an effector to detain BAG7 in the endoplasmic reticulum.

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.

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light-dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death-promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing‐based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant–pathogen interactions in gymnosperm forest trees, including kauri., RXLR effectors of the kauri dieback pathogen Phytophthora agathidicida triggered or suppressed immunity in Nicotiana, showing that RXLRs from a gymnosperm pathogen can interact with an angiosperm immune system.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen., Ustilaginoidea virens is a fungal pathogen that infects rice via the panicles. Here, the authors show that U. virens SGP1, a conserved Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored protein, elicits immune responses in rice leaves while contributing to virulence in panicles.

Significance Plant pathogens, such as Irish potato famine agent Phytophthora infestans, remain the most significant threats to global food security. Plant disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune sensors that recognize and eliminate pathogens. However, little is known about how pathogen-activated NLR immunity is influenced by environmental factors such as light. Here, we show that P. infestans manipulation of plant chloroplast function triggers a light-dependent immune response. Our results reveal that light-induced alternative promoter selection regulates plant immune recognition of a pathogen virulence factor by an agriculturally important NLR-type receptor protein., In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1–mediated resistance is compromised because plants produce a shorter GLYK—lacking the intact chloroplast transit peptide—that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root and stem rot. In this study, we developed seven pairs of polymerase chain reaction primers derived from sequence-characterized amplified regions (SCAR). These seven SCAR markers allowed discrimination of P. sojae from 17 different Phytophthora species and three other soilborne pathogens (Pythium ultimum, Fusarium solani and Rhizoctonia sp.) which also induce root rot in soybean. Among those 17 Phytophthora species, P. melonis has approximately 98% similarity in ITS sequences; P. drechsleri requires an annealing temperature up to 66 °C with an ITS-targeting diagnostic marker (PS primers) developed by Wang et al. (2006) for P. sojae; and P. sansomeana is a newly described soybean-infecting Phytophthora species. These three Phytophthora species could be specifically distinguished against P. sojae by these seven SCAR markers. After screening 100 random amplified polymorphic DNA (RAPD) primers, eight primers clearly produced specific bands only for P. sojae rather than other Phytophthora species tested. Subsequently, seven of eight P. sojae-specific RAPD markers were successfully converted into SCAR markers, namely, Scar276, Scar304, Scar333, Scar37, Scar519, Scar57 and Scar78. These SCAR markers were used to detect 75 isolates of P. sojae specifically, while no products were obtained for 29 additional isolates representing 17 other Phytophthora species and three other soilborne pathogens. Furthermore, Scar333 successfully allowed the detection with a sensitivity of 100 pg from genomic DNA of P. sojae, Scar276 had a higher sensitivity of 10 pg, and four specific SCAR primers (Scar304, Scar37, Scar519 and Scar78) had a sensitivity of 100 fg, which is the highest for detecting P. sojae until now. Six of the seven SCAR markers, with the exception of Scar57, were also used to detected P. sojae in artificial or naturally infected soybean seedlings and infested soil. Our findings demonstrate that SCAR markers provide a rapid and sensitive molecular diagnostic tool for the detection of P. sojae in plants, and will play a key role in effective management of the disease.

Due to the monocultural basis of agricultural crops, mutated plant microbes with increased pathogenicity can easily spread in the field and lead to serious yield losses. As a major threat to a wide range of crop plants, oomycete pathogens continuously undergo adaptive evolution to overcome plant defense barriers. However, the genetic basis of their evolution at the molecular level remains largely unknown. Here, we investigated the nature of variation and the population genomics of the soybean pathogen Phytophthora sojae by high-throughput genome re-sequencing. Genomic variation analysis revealed uneven “two-speed” evolutionary pattern with genes in gene-sparse regions (GSRs) showing higher rates of structural polymorphisms and positive selection. GSRs are enriched in effector genes and transposase-related genes. Our results also suggested that the NADH oxidase and MIP transporter gene families undergo rapid and diversifying selection. Furthermore, we demonstrated that P. sojae isolates possess varying numbers of RxLR effectors with diverse sequences, totaling 471 members. Among them, 42 core RxLR effectors are assumed to be indispensable for infection. Finally, we observed that Avr genes exhibit abundant sequence variation in P. sojae isolates. Several novel variants lead to the evading of host resistance, including a complete deletion in Avr3c and amino acid mutations in Avr1a. Taken together, our results provide an adaptive landscape of P. sojae at single-nucleotide resolution, as well as resources for further studies of resistance breeding and disease prevention against this important plant pathogen.

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.

Alternative splicing (AS) of pre-mRNAs increases transcriptome and proteome diversity, regulates gene expression through multiple mechanisms, and plays important roles in plant development and stress responses. However, the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly understood. Here, we examined the global AS changes in tomato leaves infected with Phytophthora infestans, the infamous Irish famine pathogen. We show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed, indicating that AS is a distinct layer of transcriptome reprogramming during plant–pathogen interactions. Furthermore, our results show that P. infestans subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility factors. To study the underlying mechanism, we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant AS. We identified nine splicing regulatory effectors (SREs) from 87 P. infestans effectors. Further studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant immunity. Our study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens, providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity.

Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance., The role of most plant leucine-rich repeat (LRR) receptors in innate immunity is unknown. Here, the authors develop virus-based constructs to silence LRR receptor-like genes in the Nicotiana benthamiana genome and identify Response to XEG1 that specifically recognizes the glycoside hydrolase 12 protein XEG1.

The apoplastic space between the plant cell wall and the plasma membrane constitutes a major battleground for plant-pathogen interactions. To survive in harsh conditions in the plant apoplast, pathogens must cope with various immune responses. During infection, plant pathogens secrete an arsenal of effector proteins into the apoplast milieu, some of which are detected by the plant surveillance system and, thus, activate plant innate immunity. Effectors that evade plant perception act in modulating plant apoplast immunity to favor successful pathogen infection. The concerted actions of apoplastic effectors often determine the outcomes of plant-pathogen interactions. In this review, we summarize current advances on the understanding of apoplastic effectors and highlight the strategies employed by pathogens to counter host apoplastic defense.

In September 2020, a disease resembling stem blight on soybean was found in Chengdu city, Sichuan province, southwestern of China. Symptoms began as a brown lesion on the stems, usually at the nodes, then lesions expanded, darkened, and even girdled the stems, causing wilt of the above stems (Figure 1A). In three 0.33-ha fields, a total of 300 soybean plants (20 plants/site × 5 sites/filed × 3 fields) were investigated, and 3% of the plants showed the disease symptoms. The symptoms were consistent with those previously reported for stem canker and stem blight on soybean caused by Diaporthe complex (Cui et al. 2009; Mena et al. 2019; Santos et al. 2011). The tissues of symptomatic soybean stems were rinsed by water, disinfected by submerging them in 75% ethanol for 30 s and in 2% sodium hypochlorite solution for 2 min, then followed by washing with sterile distilled water. Small diseased tissue fragments were placed on selective potato dextrose agar (PDA) containing rifampicin and ampicillin (both 50 mg/μl). Plates were sealed and incubated at 26°C for two days. Developing mycelia of isolates were transferred to fresh PDA and purified by single hyphal tip. For the five obtained isolates (Figure 1B), five markers, including the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, parts of the translation elongation factor 1-α (TEF1), part of the histone H3 (HIS) gene, the calmodulin gene (CAL), and the beta-tubulin gene (TUB), were amplified using the established primers ITS4/ITS5 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F (Crous et al. 2006) and H3-1b (Glass and Donaldson 1995), CAL228F/CAL737R (Carbone and Kohn 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995), respectively, and sequenced (GenBank IDs: MW595761-MW595780 and MW624472-MW624476). Phylogenetic trees were constructed based on concatenated sequences of the five markers using the maximum-likelihood (ML) method in MEGA-5.2.2. Based the results of morphological (Figure 1C-E) and phylogenetic analysis (Figure 2), the five isolates were all identified as D. phaseolorum. Pathogenicity tests for the isolates were conducted on 7-day-old soybean seedlings (cv. Shangdou 1310) using a hypocotyl slit inoculation method. At the stem 2-cm below cotyledon, a 6-mm long slit was cut with a sterile scalpel, and placed inside with a 3 mm × 3 mm PDA plug with or without mycelia of pathogen. Ten plants were assayed for each treatment, and the plants were maintained in greenhouse at 26°C, with humidity higher than 90% for the first two days. The assay was repeated at least three times. Typical brown lesions on the stems were observed four days after inoculation (Figure 1F), even 20% treated plants died. The D. phaseolorum was reisolated from these stem lesions. No disease symptom was observed on control plants (Figure 1G). Thus D. phaseolorum was the pathogen causing the soybean stem blight in field. To our knowledge, this is the first report of D. phaseolorum causing this disease in Sichuan province, China. The result may provide useful information for soybean disease control in this region of China. The authors declare no conflict of interest. Funding: China Agriculture Research System (CARS-004-PS14).

A loop-mediated isothermal amplification (LAMP) assay that directly detects Colletotrichum truncatum in diseased soybean tissues is described, thus allowing rapid diagnosis of soybean anthracnose. Using the target gene Rpb1 (that codes for the large subunit of RNA polymerase II), we designed and screened a set of species-specific primers allowing amplification at 62 °C over 70 min. After addition of SYBR Green I to the LAMP reaction products, a yellow-green color (visible to the unaided eye) developed only in the presence of C. truncatum. The detection limit of the LAMP assay was 100 pg (per μL genomic DNA). The Rpb1-Ct-LAMP assay has been successfully used to diagnose soybean anthracnose in field samples collected from Jiangsu, Anhui and Hubei provinces of China, and to detect C. truncatum in soybean seeds from farmers’ markets. Our results show that the Rpb1-Ct-LAMP assay is a useful and convenient method for detecting C. truncatum, and thus for diagnosis of soybean anthracnose.

Due to the monocultural basis of agricultural crops, mutated plant microbes with increased pathogenicity can easily spread in the field and lead to serious yield losses. As a major threat to a wide range of crop plants, oomycete pathogens continuously undergo adaptive evolution to overcome plant defense barriers. However, the genetic basis of their evolution at the molecular level remains largely unknown. Here, we investigated the nature variation and the population genomics of the soybean pathogen Phytophthora sojae by high-throughput genome re-sequencing. Genomic variation analysis revealed uneven “two-speed” evolutionary pattern with genes in gene-sparse regions (GSRs) showing higher rates of structural polymorphisms and positive selection. GSRs are enriched in effector genes and transposase-related genes. Our results also suggested that the NADH oxidase and MIP transporter gene families undergo rapid and diversifying selection. Furthermore, we demonstrated that P. sojae isolates possess varying numbers of RxLR effectors with diverse sequences, totaling 471 members. Among them, 42 core RxLR effectors are assumed to be important for infection. Finally, we observed that Avr genes exhibit abundant sequence variation in P. sojae isolates. Several novel variants lead to the evading of host resistance, including a complete deletion in Avr3c and amino acid mutations in Avr1a. Taken together, our results provide an adaptive landscape of P. sojae at single-nucleotide resolution, as well as resources for further resistance breeding and disease prevention against this important plant pathogen.

Returning straw to soil is an effective way to sustain or improve soil quality and crop yields. However, a robust understanding of the impact of straw return on the composition of the soil microbial communities under field conditions has remained elusive. In this study, we characterized the effects of wheat straw return on soil bacterial and fungal communities in a wheat–soybean rotation system over a 3-year period, using Illumina-based 16S rRNA, and internal transcribed region (ITS) amplicon sequencing. Wheat straw return significantly affected the α-diversity of the soil bacterial, but not fungal, community. It enhanced the relative abundance of the bacterial phylum Proteobacteria and the fungal phylum Zygomycota, but reduced that of the bacterial phylum Acidobacteria, and the fungal phylum Ascomycota. Notably, it enriched the relative abundance of nitrogen-cycling bacterial genera such as Bradyrhizobium and Rhizobium. Preliminary analysis of soil chemical properties indicated that straw return soils had significantly higher total nitrogen (TN) contents than no straw return soils. In addition, the relative abundance of fungal genera containing pathogens was significantly lower in straw return soils relative to control soils, such as Fusarium, Alternaria, and Myrothecium. These results suggested a selection effect from the 3-year continuous straw return treatment and the soil bacterial and fungal communities were moderately changed.

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae ( Ps PSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of Ps PSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the Ps PSR2-like arrangement, that is, containing a W-Y motif as the “start” unit, various numbers of L-W-Y motifs as the “middle” units, and a degenerate L-W-Y as the “end” unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of Ps PSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a “building block” to accelerate effector evolution in Phytophthora .

Phytophthora pathogens manipulate host innate immunity by secreting numerous RxLR effectors, thereby facilitating pathogen colonization. Predicted single and tandem repeats of WY domains are the most prominent C-terminal motifs conserved across the Phytophthora RxLR superfamily. However, the functions of individual WY domains in effectors remain poorly understood. The Phytophthora sojae effector PSR1 promotes infection by suppressing small RNA biogenesis in plant hosts. We identified one single WY domain following the RxLR motif in PSR1. This domain was required for RNA silencing suppression activity and infection in Nicotiana benthamiana, Arabidopsis and soybean. Mutations of the conserved residues in the WY domain did not affect the subcellular localization of PSR1 but abolished its effect on plant development and resistance to viral and Phytophthora pathogens. This is at least in part due to decreased protein stability of the PSR1 mutants in planta. The identification of the WY domain in PSR1 allows predicts that a family of PSR1-like effectors also possess RNA silencing suppression activity. Mutation of the conserved residues in two members of this family, PpPSR1L from P. parasitica and PcPSR1L from P. capsici, perturbed their biological functions, indicating that the WY domain is critical in Phytophthora PSR1 and PSR1-like effectors.

Phytophthora sansomeana E.M. Hansen & Reeser is a newly described species and infects Douglas-fir, alfalfa, and soybean (1). Soybean production is an important part of the local economy in Yili State in Xinjiang Uygur Autonomous Region, northwest China. Unfortunately since 2005, root and stem rot disease has emerged on a number of farms. To identify the causal agent, plant samples with symptoms, including whole plant wilting or yellowing and stunting, were collected from fields during 2005 and 2008. Tissue from the edges of stem lesions was placed on selective lima bean agar (LBA) at 20°C for 3 to 4 days (2,3). Four single zoospore isolates of Phytophthora were obtained and maintained on LBA or 10% V8 juice liquid medium for examination of morphological and physiological characteristics. The colonies on LBA were aerial and rosaceous. The isolates were homothallic, and oogonia and oospores were readily produced in culture after 7 days on LBA plates. Oogonia averaged 38 μm and oospore width ranged from 23 to 48 μm and averaged 31 μm. Antheridia were approximately 15 × 12 μm and predominantly amphigynous in V8 juice. Sporangia were terminal or paragynous on persistent sporangiophores, nonpapillate, ovoid to obpyriform, and measured 52 × 35 μm with an average length/breadth ratio of 1.5. Hyphal swellings were produced in V8 juice 2 days after inoculation. The optimum temperature for growth was approximately 25°C and none occurred at 0 or 35°C. The internal transcribed spacer (ITS) sequence of this Phytophthora species (GenBank FJ966880) agreed 100% with sequences of P. sansomeana isolates deposited in GenBank (GQ853880 and EU925375). Pathogenicity tests were performed by hypocotyl inoculation method (2) using isolate Yili71 and potted soybean cv. Williams. Plants were grown in a growth chamber for 10 days before inoculation in 16-cm-diameter pots (2). Plants were inoculated with 2- × 2-mm plugs of mycelium grown for 4 days on LBA at 25°C, the plugs were adhered to the sides of wounded lower hypocotyls. As controls, plants were inoculated with LBA agar plugs without mycelium (2). Inoculated plants were maintained in a growth chamber at approximately 25°C with a 10-h dark/14-h light cycle and 50% relative humidity and symptom development was monitored daily for 1 week. Wounded stems inoculated with mycelium developed water-soaked lesions, which were similar to those seen on naturally infected plants. A Phytophthora sp. was reisolated from the margins of expanding lesions on wounded stems. To our knowledge, this is the first report of P. sansomeana infection of soybean in China and the threat it may pose to soybean production is unclear. References: (1) E. M. Hansen et al. Mycologia 101:129. 2009. (2) Z. Y. Wang et al. Fungal Genet. Biol. 43:826, 2006. (3) X. B. Zheng. Methods in Phytophthora. Chinese Agriculture Press. Beijing, China, 1995.

Phytophthora sojae causes root and stem rot, one of the most devastating diseases of soybean worldwide. In Heilongjiang and Fujian provinces in China, serious cases of Phytophthora stem and root rot have occurred and caused heavy losses in the past several years. To determine the current population status of this pathogen, we investigated the pathogen's distribution, pathotypes, and metalaxyl sensitivity in both provinces. P. sojae was baited and isolated from 258 soil samples in both provinces using the soybean leaf bait method. The pathotypes of all isolates were characterized on 13 differential soybean cultivars using the hypocotyl slit inoculation method, and the sensitivity of all isolates to metalaxyl was tested in vitro. In all, 75 isolates were recovered from 75 fields in 33 counties; of these, 31 counties were in Heilongjiang Province and 2 counties were in Fujian Province. Thirty-five new pathotypes were identified compared with the previously defined races. Less than 5% of the isolates were virulent to cultivars with individual Rps genes 1a, 1c, or 1k. No metalaxyl-resistant isolates were found; the half maximal effective concentration values of all isolates ranged from 0.04 to 0.22 μg ml–1. These results suggest that effective management of the disease in both provinces can be accomplished through the use of resistant cultivars with Rps genes 1a, 1c, or 1k and the fungicide metalaxyl.

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., eLife digest Just like animals, plants can become infected and diseased. Among the microbes that infect plants, one group tends to stand out. Named Phytophthora after the Greek for ‘the plant destroyer’, these fungus-like microbes cause diseases in many species of plant, including important food crops. These diseases are difficult to control, and as a result Phytophthora diseases cost the farming industry billions of dollars every year. Effective control of Phytophthora diseases is likely to depend on scientists first gaining a better understanding of how these microbes infect plants. Also like animals, plants have an immune system to protect themselves from disease. Yet many disease-causing microbes make so-called effector proteins to overcome their hosts’ defenses. Previously in 2015, researchers reported that one effector made by a species known as Phytophthora sojae could suppress the immune system of soybean plants during the early stages of an infection. But it was not clear how the effector achieved this. Now, Li et al., who include many of the researchers involved in the 2015 study, go on to show that the same effector, known as PsAvh52, helps P. sojae to infect soybean plants by interacting with a previously unknown soybean enzyme. The enzyme is a transacetylase, meaning it belongs to a group of enzymes that transfer a chemical marker called an acetyl group on to other molecules including proteins. Li et al. went on to show that the PsAvh52 effector essentially hijacks the transacetylase enzyme, moving it to a location in the cell nucleus where it could chemically modify the proteins that package the soybean plant’s DNA. These chemical changes activate nearby genes that would have otherwise been switched off, and these incorrectly activated genes make the plant more susceptible to the infection. By deciphering one of the strategies that helps P. sojae to infect soybean plants, Li et al. have uncovered two possible approaches that may help to get this plant disease under control. The findings highlight the effector PsAvh52 as a weapon that could be blocked; they also reveal the transacetylase enzyme as a vulnerable point in the plant that could be protected. The next step will be to explore if there are chemical or genetic means that can achieve either of these two goals.

On the basis of its downy mildew–like morphology, the litchi downy blight pathogen was previously named Peronophythora litchii. Recently, however, it was proposed to transfer this pathogen to Phytophthora clade 4. To better characterize this unusual oomycete species and important fruit pathogen, we obtained the genome sequence of Phytophthora litchii and compared it to those from other oomycete species. P. litchii has a small genome with tightly spaced genes. On the basis of a multilocus phylogenetic analysis, the placement of P. litchii in the genus Phytophthora is strongly supported. Effector proteins predicted included 245 RxLR, 30 necrosis-and-ethylene-inducing protein-like, and 14 crinkler proteins. The typical motifs, phylogenies, and activities of these effectors were typical for a Phytophthora species. However, like the genome features of the analyzed downy mildews, P. litchii exhibited a streamlined genome with a relatively small number of genes in both core and species-specific protein families. The low GC content and slight codon preferences of P. litchii sequences were similar to those of the analyzed downy mildews and a subset of Phytophthora species. Taken together, these observations suggest that P. litchii is a Phytophthora pathogen that is in the process of acquiring downy mildew–like genomic and morphological features. Thus P. litchii may provide a novel model for investigating morphological development and genomic adaptation in oomycete pathogens.

Ansary M.W.R., Haque E., West H.M., Rahman M.M., Akanda A.M., Wang Y., Islam M. T. (2016): Medicinal plant extracts and protein kinase C inhibitor suppress zoosporogenesis and impair motility of Phytophthora capsici zoospores. Plant Protect. Sci., 52: 113–122. The effects of water and acetone extracts from 100 medicinal plants growing in Bangladesh, along with a selective inhibitor of protein kinase C (PKC), chelerythrine chloride, were tested on zoosporogenesis (release of zoospores from the sporangia) and motility of Phytophthora capsici zoospores. Among 10 active crude acetone extracts, those from Psidium guajava and Nigella sativa (100 μg/ml) suppressed zoosporogenesis relative to the control (100% zoospore release) to 60 and 40% released, respectively and inhibited motility of 100% of the zoospores within 60 min of treat ment. Chelerythrine chloride also suppressed zoosporogenesis (30% released) at 0.1 μg/ml and inhibited motility of 100% zoospores at 0.2 μg/ml within 60 minutes. Among water extracts of 100 medicinal plants, 56 impaired motility of zoospores in a dose- and time-dependent manner. Diluted (20-fold) water extracts of 10 plants including Ocinum gratissimum, Terminalia bohera , and Duranta plumeri inhibited motility and subsequently caused lysis of zoospores.

A new method of direct detection of pathogenic fungi in infected soybean tissues has been reported here. The method rapidly diagnoses soybean seedling blight caused by Rhizoctonia solani and soybean charcoal rot caused by Macrophomina phaseolina, and features loop-mediated isothermal amplification (LAMP). The primers were designed and screened using internal transcribed spacers (ITS) as target DNAs of both pathogens. An ITS-Rs-LAMP assay for R. solani and an ITS-Mp-LAMP assay for M. phaseolina that can detect the pathogen in diseased soybean tissues in the field have been developed. Both LAMP assays efficiently amplified the target genes over 60 min at 62°C. A yellow-green color (visible to the naked eye) or intense green fluorescence (visible under ultraviolet light) was only observed in the presence of R. solani or M. phaseolina after addition of SYBR Green I. The detection limit of the ITS-Rs-LAMP assay was 10 pg μl–1 of genomic DNA; and that of the ITS-Mp-LAMP assay was 100 pg μl–1 of genomic DNA. Using the two assays described here, we successfully and rapidly diagnosed suspect diseased soybean samples collected in the field from Jiangsu and Anhui provinces.

The ability of sensing the signals from hosts, aggregating, and searching for proper sites of invasion is crucial for many soil-borne plant pathogens. One of these is Phytophthora sojae that specifically infects soybean and causes severe yield loss. In the field P. sojae zoospores attracted to soybean exudates isoflavones is an essential step for early infection. Recent studies based on genome mining and gene silencing technologies have shown that this special zoospore behavior relies on G-protein mediated signaling pathways. This review is to summarize researches on P. sojae functional genomics related to G-protein signaling and briefly describe the molecular mechanism of P. sojae zoospore chemotaxis to root exudates, especially isoflavones.

Plants associate with diverse microbes that exert beneficial, neutral, or pathogenic effects inside the host. During the initial stages of invasion, the plant apoplast constitutes a hospitable environment for invading microbes, providing both water and nutrients. In response to microbial infection, a number of secreted proteins from host cells accumulate in the apoplastic space, which is related to microbial association or colonization processes. However, the molecular mechanisms underlying plant modulation of the apoplast environment and how plant-secreted proteases are involved in pathogen resistance are still poorly understood. Recently, several studies have reported the roles of apoplastic proteases in plant resistance against bacteria, fungi, and oomycetes. On the other hand, microbe-secreted proteins directly and/or indirectly inhibit host-derived apoplastic proteases to promote infection. These findings illustrate the importance of apoplastic proteases in plant–microbe interactions. Therefore, understanding the protease-mediated apoplastic battle between hosts and pathogens is of fundamental importance for understanding plant–pathogen interactions. Here, we provide an overview of plant–microbe interactions in the apoplastic space. We define the apoplast, summarize the physical and chemical properties of these structures, and discuss the roles of plant apoplastic proteases and pathogen protease inhibitors in host–microbe interactions. Challenges and future perspectives for research into protease-mediated apoplastic interactions are discussed, which may facilitate the engineering of resistant crops., The apoplast mediates the initial battle between plants and pathogens. This review summarizes the current understanding of plant–microbe interactions in the apoplastic region, with special emphasis on the role of secreted proteases in this process.

Perception of pathogen-associated molecular patterns (PAMPs) often triggers various innate immune responses in plants. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. Here we described a protocol to monitor the relative expression level of marker genes in Nicotiana benthamiana upon treatment with PAMPs. The procedure includes leaf treatment using PAMPs, total RNA isolation, cDNA synthesis, quantitative real-time PCR and data analysis. This protocol is applicable to monitor marker gene expression triggered by different PAMPs in N. benthamiana.