Search

Introduction: Metformin (MET), derived from Galega officinalis, stands as the primary first-line medication for the treatment of type 2 diabetes (T2D). Despite its well-documented benefits in mammalian cellular processes, its functions and underlying mechanisms in plants remain unclear. Objectives: This study aimed to elucidate MET’s role in inducing plant immunity and investigate the associated mechanisms. Methods: To investigate the impact of MET on enhancing plant immune responses, we conducted assays measuring defense gene expression, reactive oxygen species (ROS) accumulation, mitogen-activated protein kinase (MAPK) phosphorylation, and pathogen infection. Additionally, surface plasmon resonance (SPR) and microscale thermophoresis (MST) techniques were employed to identify MET targets. Protein-protein interactions were analyzed using a luciferase complementation assay and a co-immunoprecipitation assay. Results: Our findings revealed that MET boosts plant disease resistance by activating MAPKs, upregulating the expression of downstream defense genes, and fortifying the ROS burst. CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) was identified as a target of MET. It inhibited the interaction between BOTRYTIS-INDUCED KINASE 1 (BIK1) and CPK28, blocking CPK28 threonine 76 (T76) transphosphorylation by BIK1, and alleviating the negative regulation of immune responses by CPK28. Moreover, MET enhanced disease resistance in tomato, pepper, and soybean plants. Conclusion: Collectively, our data suggest that MET enhances plant immunity by blocking BIK1-mediated CPK28 phosphorylation.

Abstract Oomycete pathogens deliver many effectors to enhance virulence or suppress plant immunity. Plant immune networks are interconnected, in which a few effectors can trigger a strong defense response when recognized by immunity-related proteins. How effectors activate plant defense response remains poorly understood. Here we report Phytophthora capsici effector RxLR23KM can induce plant cell death and plant immunity. RxLR23KM specifically binds to ERD15La, a regulator of abscisic acid and salicylic acid pathway, and the binding intensity depends on the amino acid residues (K93 and M320). NbNAC68, a downstream protein of ERD15La, can stimulate plant immunity that is compromised after binding with ERD15La. Silencing of NbNAC68 substantially prevents the activation of plant defense response. RxLR23KM binds to ERD15La, releasing NbNAC68 to activate plant immunity. These findings highlight a strategy of plant defense response that ERD15La as a central regulator coordinates RxLR23KM to regulate NbNAC68-triggered plant immunity.

Receptor protein kinases (RPKs) critically provide the basic infrastructure to sense, perceive, and conduct the signalling events at the cell surface of organisms. The importance of LRR-RLKs has been well studied in plants, but much less information has been reported in oomycetes. In this work, we have silenced the PcLRR-RK3 and characterised its functional importance in Phytophthora capsici. PcLRR-RK3 was predicted to encode signal peptides, leucine-rich repeats, transmembrane, and kinase domains. PcLRR-RK3-silenced transformants showed impaired colony growth, decreased deformed sporangia, and reduced zoospores count. The mycelium of silenced transformants did not penetrate within the host tissues and showed defects in the pathogenicity of P. capsici. Interestingly, gene silencing also weakens the ability of zoospores germination and penetration into host tissues and fails to produce necrotic lesions. Furthermore, PcLRR-RK3 localisation was found to be the plasma membrane of the cell. Altogether, our results revealed that PcLRR-RK3 was required for the regulation of vegetative growth, zoospores penetration, and establishment into host leaf tissues.

Abstract The mitogen-activated protein kinase (MAPK) cascade MEK2-SIPK/WIPK is essential for immunity in Solanaceae plants. This cascade is tightly controlled to prevent harmful hyperactivation. However, the E3 ubiquitin ligases utilized by plants to reduce MEK2- SIPK/WIPK protein levels remain largely elusive. Here, we confirmed the essential role of Nicotiana benthamiana MEK2-SIPK/WIPK in resistance to the oomycete pathogen Phytophthora capsici. Using tobacco rattle virus (TRV)-based gene silencing, we screened prevalent plant U-box protein (PUB)-type E3 ligases with Armadillo (ARM) repeats to characterize those involved in Phytophthora resistance and MEK2-SIPK/WIPK degradation. We found that pub40 knockdown mutants exhibited significantly enhanced resistance to P. capsici. NbPUB40 was under ubiquitination and proteasomal degradation in planta, with two conserved sites (Cys28 and Val41) in the U-box domain being essential for its activity. NbPUB40 was shown to interact with the whole MEK2-SIPK/WIPK cascade and promote their degradation, the ubiquitination levels of which were also notably reduced in the pub40 mutant. Our results reveal a mechanism in which a PUB E3 ubiquitin ligase negatively regulates plant P. capsici resistance by destabilizing the MEK2-SIPK/WIPK cascade.

Abstract Plant cells perceive pathogen invasion by recognizing microbial patterns using plasma-membrane-localized pattern-recognition receptors (PRRs) to initiate pattern-triggered immunity (PTI), which confers a moderate immunity to most microbes. For instance, the PRR FLS2 (FLAGELLIN SENSING 2) recognizes bacterial flagellin in the presence of the co-receptor BAK1 and activates a series of PTI responses, such as reactive oxygen species (ROS) burst and mitogen-activated protein kinase (MAPK) activation. We previously showed that soybean malectin/malectin-like domain-containing receptor-like kinase (MRLK) protein GmLMM1 negatively regulates PTI by suppressing FLS2-BAK1 interaction. GmLMM1 replicates in tandem with five other GmMRLKs on chromosome 13. Here, we show that GmMRLK32, the closest homolog to GmLMM1 among the tandem genes of GmLMM1, negatively regulates PTI and disease resistance against bacterial and oomycete pathogens. The Gmmrlk32 mutant showed enhanced flg22-induced ROS burst and MAPK activation. We revealed that GmMRLK32 interacts with GmFLS2 and GmBAK1, and suppresses flg22-induced GmFLS2-GmBAK1 dimerization in a manner similar to that of GmLMM1. We further showed that GmMRLK32 specifically interacts with GmLMM1 to regulate PTI. In Nicotiana benthamiana plants, co-expression of GmMRLK32 and GmLMM1 showed a stronger PTI inhibitory effect on PTI activation than expression of GmMRLK32 or GmLMM1 alone. We uncovered a novel mechanism by which GmMRLK32 and GmLMM1 coordinately regulate PTI by forming hetero-oligomer.

Abstract Phytophthora are destructive plant pathogens that pose a serious threat to crop production. Traditional control methods rely heavily on chemical fungicides, which are harmful to the environment and human health. Currently, effective green prevention and control methods for Phytophthora pathogens are lacking. Plants rely primarily on their innate immune system to resist pathogens. Plant cells perceive pathogen invasion and activate immune responses by recognizing specific pathogen-derived molecules, called elicitors, which mainly include pathogen-associated molecular patterns (PAMPs) and microbial effector proteins. PAMPs, which are conserved molecular features of microbes and recognized by plant cell surface-localized pattern-recognition receptors (PRRs), activate mild and broad-spectrum disease resistance. However, there are few reports on elicitor proteins that induce broad resistance against Phytophthora pathogens. In this study, we identified BcIEB1, a fungal-derived PAMP, which activated plant immune responses in a BAK1- and SOBIR1-dependent manner. BcIEB1 could induce plant resistance to various Phytophthora pathogens, including P. capsici, P. infestans, and P. parasitica. We further found that the combination of lower concentrations of BcIEB1 with fungicides, such as pyraclostrobin, azoxystrobin, and metalaxyl-M, could enhance the effect on Phytophthora disease control while reducing the dependence on fungicides, thereby reducing environmental pollution. This study identified a novel, less toxic strategy for controlling Phytophthora diseases.

Abstract Plants have evolved a multilayered and sophisticated immune system to establish effective resistance to a variety of pathogens. Phytophthora species are among the most notorious plant pathogens, causing destructive diseases on a variety of agricultural crops. Understanding the plant immune system is crucial for protecting crops from Phytophthora diseases. Here, we summarize the recent work on genes involved in plant resistance against Phytophthora pathogens, including cell surface pattern recognition receptors, cytoplasmic nucleotide-binding leucine-rich repeat receptors, regulator genes, and non-host resistance genes, small RNA, and long non-coding RNA are also discussed in this review. Although the molecular mechanisms of only a small proportion of them have been clarified, emergence of new mechanisms of plant defense will offer exciting opportunities for utilization of these genes in disease resistance breeding as well as generation of disease-resistant crop germplasms.

Abstract Background Potato late blight, caused by Phytophthora infestans, is the most devastating disease on potato. Dissecting critical immune components in potato will be supportive for engineering P. infestans resistance. Upon pathogens attack, plant Ca2+ signature is generated and decoded by an array of Ca2+ sensors, among which calcineurin B-like proteins (CBLs) coupled with plant specific CBL-interacting protein kinases (CIPKs) are much less explored in plant immunity. Results In this study, we identified that two differential potato CBL-CIPK modules regulate plant defense responses against Phytophthora and ROS production, respectively. By deploying virus-induced gene silencing (VIGS) system-based pathogen inoculation assays, StCBL3 was shown to negatively regulate Phytophthora resistance. Consistently, StCBL3 was further found to negatively regulate PTI and ETI responses in Nicotiana benthamiana. Furthermore, StCIPK7 was identified to act together with StCBL3 to negatively regulate Phytophthora resistance. StCIPK7 physically interacts with StCBL3 and phosphorylates StCBL3 in a Ca2+-dependent manner. StCBL3 promotes StCIPK7 kinase activity. On the other hand, another StCBL3-interacting kinase StCIPK24 negatively modulating flg22-triggered accumulation of reactive oxygen species (ROS) by interacting with StRBOHB. Conclusions Together, these findings demonstrate that the StCBL3-StCIPK7 complex negatively modulates Phytophthora resistance and StCBL3-StCIPK24 complex negatively regulate ROS production. Our results offer new insights into the roles of potato CBL-CIPK in plant immunity and provide valuable gene resources to engineer the disease resistance potato in the future.

Oomycete pathogens secrete numerous crinkling and necrosis proteins (CRNs) to manipulate plant immunity and promote infection. However, the functional mechanism of CRN effectors is still poorly understood. Previous research has shown that the Phytophthora sojae effector PsCRN108 binds to the promoter of HSP90s and inhibits their expression, resulting in impaired plant immunity. In this study, we found that in addition to HSP90, PsCRN108 also suppressed other Heat Shock Protein (HSP) family genes, including HSP40. Interestingly, PsCRN108 inhibited the expression of NbHSP40 through its promoter, but did not directly bind to its promoter. Instead, PsCRN108 interacted with NbCAMTA2, a negative regulator of plant immunity. NbCAMTA2 was a negative regulator of NbHSP40 expression, and PsCRN108 could promote such inhibition activity of NbCAMTA2. Our results elucidated the multiple roles of PsCRN108 in the suppression of plant immunity and revealed a new mechanism by which the CRN effector hijacked transcription factors to affect immunity. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Potato late blight caused by Phytophthora infestans is a devastating disease worldwide. Unlike other plant pathogens, double-stranded RNA (dsRNA) is poorly taken up by P. infestans, which is a key obstacle in using dsRNA for disease control. Here, a self-assembled multicomponent nano-bioprotectant for potato late blight management is designed based on dsRNA and a plant elicitor. Nanotechnology overcomes the dsRNA delivery bottleneck for P. infestans and extends the RNAi protective window. The protective effect of nano-enabled dsRNA against infection arises from a synergistic mechanism that bolsters the stability of dsRNA and optimizes its effective intracellular delivery. Additionally, the nano-enabled elicitor enhances endocytosis and amplifies the systemic defense response of the plants. Co-delivery of dsRNA and an elicitor provides a protective effect via the two aspects of pathogen inhibition and elevated plant defense mechanisms. The multicomponent nano-bioprotectant exhibits superior control efficacy compared to a commercial synthetic pesticide in field conditions. This work proposes an eco-friendly strategy to manage devastating plant diseases and pests.

Abstract Public metabolites such as vitamins play critical roles in maintaining the ecological functions of microbial community. However, the biochemical and physiological bases for fine-tuning of public metabolites in the microbiome remain poorly understood. Here, we examine the interactions between myxobacteria and Phytophthora sojae, an oomycete pathogen of soybean. We find that host plant and soil microbes complement P. sojae’s auxotrophy for thiamine. Whereas, myxobacteria inhibits Phytophthora growth by a thiaminase I CcThi1 secreted into extracellular environment via outer membrane vesicles (OMVs). CcThi1 scavenges the required thiamine and thus arrests the thiamine sharing behavior of P. sojae from the supplier, which interferes with amino acid metabolism and expression of pathogenic effectors, probably leading to impairment of P. sojae growth and pathogenicity. Moreover, myxobacteria and CcThi1 are highly effective in regulating the thiamine levels in soil, which is correlated with the incidence of soybean Phytophthora root rot. Our findings unravel a novel ecological tactic employed by myxobacteria to maintain the interspecific equilibrium in soil microbial community.

Abstract Plant cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) and receptor-like proteins (LRR-RLPs) form dynamic complexes to receive a variety of extracellular signals. LRR-RLKs are also widespread in oomycete pathogens, whereas it remains enigmatic whether plant and oomycete LRR-RLKs could mediate cell-to-cell communications between pathogen and host. Here, we report that an LRR-RLK from the soybean root and stem rot pathogen Phytophthora sojae, PsRLK6, can activate typical pattern-triggered immunity in host soybean and nonhost tomato and Nicotiana benthamiana plants. PsRLK6 homologs are conserved in oomycetes and also exhibit immunity-inducing activity. A small region (LRR5-6) in the extracellular domain of PsRLK6 is sufficient to activate BAK1- and SOBIR1-dependent immune responses, suggesting that PsRLK6 is likely recognized by a plant LRR-RLP. Moreover, PsRLK6 is shown to be up-regulated during oospore maturation and essential for the oospore development of P. sojae. Our data provide a novel type of microbe-associated molecular pattern that functions in the sexual reproduction of oomycete, and a scenario in which a pathogen LRR-RLK could be sensed by a plant LRR-RLP to mount plant immunity.

Yam (Dioscorea spp.) is an important tuber crop consumed globally. However, stable yam production faces challenges from a variety of diseases caused by fungi, nematodes, viruses, and bacteria. Prominent diseases such as anthracnose, leaf spot, yam wilt, dry rot, and crazy root syndrome, currently pose serious threats to yam yields. These diseases not only result in quality degradation but also cause great economic losses. This review summarizes the damages, symptoms, causal agents, and epidemic factors of major yam diseases. It also outlines a comprehensive disease control strategy that includes the use of resistant varieties, proper crop rotation, sanitation measures, and the application of agrochemicals and biocontrol agents. Additionally, this review addresses future perspectives on risk factors and knowledge gaps, aiming to serve as a reference for in-depth research into advanced disease monitoring and control technologies for yams.

Abstract Plant pathogens cause severe losses to crop yields and economic returns in agriculture. Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics, such lab-based methods can be time-consuming and arduous to complete many samples. To mitigate these challenges, we developed an inexpensive portable DNA extraction technique that is lightweight and suitable for deployment in sampling locations, such as fields. It includes a DNA extraction device fabricated with a Steel Microneedle Array (SMA) and a simple high-efficiency DNA extraction buffer. As a result, DNA extraction times can be reduced to within ~ 1 min, and the eluted DNA is demonstrated to be suitable for subsequent molecular biological analyses without requiring additional purification. Cross-priming amplification (CPA) technology was first established to detect Phytophthora infestans, which achieves sensitivity attainment of 10–7 ng/µL. The detection result can be conveniently estimated with naked-eye visual inspection using fluorescent dsDNA binding dye. CPA was demonstrated to be more feasible than PCR-based approaches and performed well in species-specific and practicability tests. This study elucidates a novel integrated pathogen detection technique coupled with SMA-Device extraction and a modified visual CPA assay to establish and verify various field-based samples infected with multiple pathogens. Altogether, the total sample-to-answer time for pathogen detection was reduced to ~ 1.5 h, making field-based analysis affordable and achievable for farmers or extension workers inside and outside the laboratory.

Abstract Fungal pathogens are among the main destructive microorganisms for crops and ecosystems worldwide, causing substantial agricultural and economic losses. Plant cell surface-localized lysin motif (LysM)-containing receptor-like kinases (RLKs) or receptor-like proteins (RLPs) enhance plant resistance to fungal pathogens via sensing chitin, which is a conserved component of the fungal cell wall. Other types of RLKs also regulate chitin signaling via distinct mechanisms in plants. In this study, we identified a G-type lectin RLK, NbERK1, which positively regulated chitin signaling and resistance to the fungal pathogen Sclerotinia sclerotiorum in the model plant Nicotiana benthamiana. In addition, the LysM-RLK NbCERK1/NbLYK4 was shown to mediate plant resistance to S. sclerotiorum positively. Further, the association of chitin-induced NbCERK1-NbLYK4 was found to be essential for chitin perception and signaling. Importantly, NbERK1 was associated with NbCERK1/NbLYK4 and positively regulated chitin-induced NbCERK1-NbLYK4 association. Moreover, chitin could induce the dissociation of NbERK1 from the NbCERK1-NbLYK4 complex. Also, the kinase activity of NbERK1 was likely essential for this dissociation and plant resistance-enhancing activity of NbERK1. Together, these results suggest that NbERK1 is a novel component of the chitin receptor complex and enhances plant resistance to fungal pathogens via regulating chitin signaling.

The oomycete Pythium myriotylum is a necrotrophic pathogen that infects many crop species worldwide, including ginger, soybean, tomato, and tobacco. Here, we identified a P. myriotylum small cysteine-rich protein, PmSCR1, that induces cell death in Nicotiana benthamiana by screening small, secreted proteins that were induced during infection of ginger and did not have a predicted function at the time of selection. Orthologs of PmSCR1 were found in other Pythium species, but these did not have cell death–inducing activity in N. benthamiana. PmSCR1 encodes a protein containing an auxiliary activity 17 family domain and triggers multiple immune responses in host plants. The elicitor function of PmSCR1 appears to be independent of enzymatic activity, because the heat inactivation of PmSCR1 protein did not affect PmSCR1-induced cell death or other defense responses. The elicitor function of PmSCR1 was also independent of BAK1 and SOBIR1. Furthermore, a small region of the protein, PmSCR186-211, is sufficient for inducing cell death. A pretreatment using the full-length PmSCR1 protein promoted the resistance of soybean and N. benthamiana to Phytophthora sojae and Phytophthora capsici infection, respectively. These results reveal that PmSCR1 is a novel elicitor from P. myriotylum, which exhibits plant immunity-inducing activity in multiple host plants. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transmembrane kinases (TMKs) are important mediators of cellular signaling cascades. The kinase domains of most metazoan and plant TMKs belong to the serine/threonine/tyrosine kinase (S/T/Y-kinase) superfamily. They share a common origin with prokaryotic kinases and have diversified into distinct subfamilies. Diverse members of the eukaryotic crown radiation such as amoebae, ciliates, and red and brown algae (grouped here under the umbrella term “protists”) have long diverged from higher eukaryotes since their ancient common ancestry, making them ideal organisms for studying TMK evolution. Here, we developed an accurate and high-throughput pipeline to predict TMKomes in cellular organisms. Cross-kingdom analyses revealed distinct features of TMKomes in each grouping. Two-transmembrane histidine kinases constitute the main TMKomes of bacteria, while metazoans, plants, and most protists have a large proportion of single-pass TM S/T/Y-kinases. Phylogenetic analyses classified most protist S/T/Y-kinases into three clades, with clades II and III specifically expanded in amoebae and oomycetes, respectively. In contrast, clade I kinases were widespread in all protists examined here, and likely shared a common origin with other eukaryotic S/T/Y-kinases. Functional annotation further showed that most non-kinase domains were grouping-specific, suggesting that their recombination with the more conserved kinase domains led to the divergence of S/T/Y-kinases. However, we also found that protist leucine-rich repeat (LRR)- and G-protein-coupled receptor (GPCR)-type TMKs shared similar sensory domain architectures with respective plant and animal TMKs, despite that they belong to distinct kinase subfamilies. Collectively, our study revealed the functional diversity of TMKomes and the distinct origins of S/T/Y-kinases in protists.

Summary: Protein homeostasis is vital for organisms and requires chaperones like the conserved Bcl-2-associated athanogene (BAG) co-chaperones that bind to the heat shock protein 70 (HSP70) through their C-terminal BAG domain (BD). Here, we show an unconventional BAG subfamily exclusively found in oomycetes. Oomycete BAGs feature an atypical N-terminal BD with a short and oomycete-specific α1 helix (α1′), plus a C-terminal small heat shock protein (sHSP) domain. In oomycete pathogen Phytophthora sojae, both BD-α1′ and sHSP domains are required for P. sojae BAG (PsBAG) function in cyst germination, pathogenicity, and unfolded protein response assisting in 26S proteasome-mediated degradation of misfolded proteins. PsBAGs form homo- and heterodimers through their unique BD-α1′ to function properly, with no recruitment of HSP70s to form the common BAG-HSP70 complex found in other eukaryotes. Our study highlights an oomycete-exclusive protein homeostasis mechanism mediated by atypical BAGs, which provides a potential target for oomycete disease control.

Abstract Heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are key regulators of eukaryotic intracellular signaling. Extra-large G (XLG) proteins are a subfamily of plant-specific Gα proteins interacting with plasma membrane-localized receptors to regulate multiple biological processes. The Nicotiana benthamiana genome encodes seven XLG proteins, NbXLG1–7, whose functions in disease resistance and underlying mechanisms are unknown. In this study, we silenced all the seven genes and found that disease susceptibility was enhanced when both NbXLG3 and NbXLG5 or NbXLG4 was silenced. Then, we generated N. benthamiana xlg3xlg5 double- and xlg4 single-mutant lines using the CRISPR-Cas9 approach. All the mutants showed reduced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, the fungal pathogen Sclerotinia sclerotiorum, and a series of oomycete pathogens, including Phytophthora capsici, Phytophthora infestans, and Phytophthora parasitica. We further demonstrated that NbXLG3/4/5 positively regulated microbial pattern-induced reactive oxygen species burst and defense gene expression by directly coupling to the tested plant immune receptors. In addition, we examined the role of NbXLG3/4/5 in abiotic stress tolerance and observed that NbXLG3 and NbXLG5 negatively regulated plant resistance to high-salt, mannitol, and PEG. Our study demonstrates the possible role of NbXLG3/4/5 in response to biotic and abiotic stresses and provides insights for the improvement of plant resistance to environmental changes.

Abstract Plant immunity is activated upon perception of pathogens and often affects growth when it is constitutively active. It is still a challenge to balance plant immunity and growth in disease resistance breeding. Here, we demonstrated that soybean (Glycine max) polyamine oxidase (GmPAO) confers resistance to multiple Phytophthora pathogens, but has no obvious adverse impact on agronomic traits. GmPAO produces H2O2 by oxidizing spermidine and spermine. Phytophthora sojae induces an increase in these two substrates, and thus promotes GmPAO-mediated polyamine catabolism specifically during infection. Interestingly, we found that the two substrates showed higher accumulation in transgenic soybean lines overexpressing GmPAO than in WT and CK after inoculation with P. sojae to ensure H2O2 production during infection, rather than directly inhibit P. sojae. In these transgenic soybean plants, the significantly enhanced resistance to different P. sojae isolates was achieved; PAMP-induced H2O2 accumulation was enhanced by GmPAO overexpression. Moreover, transient expression of GmPAO also significantly improved Nicotiana benthamiana resistance to Phytophthora capsici and Phytophthora parasitica in agroinfiltration assays. Our results provide a novel approach to allow rapid defense responses in plants upon pathogen infection while minimizing growth penalties under normal conditions, with a clear mechanism in which plant promotes H2O2 production via pathogen-activated substrates.

Abstract Plant pathogenic oomycete species pose a worldwide threat to crop production and ecosystems. During infection, oomycete pathogens secrete a series of effectors to manipulate plant immunity. Many of these effectors, which are indicated as avirluence gene candidates, will use components of immunity pathway to induce cell death in plants. This response given by plants is known as effector-triggered immunity (ETI). The identification of avirulence genes from pathogenic oomycete species opens a way to investigating their virulence function and uncovering related R gene repertoires in resistant plants. In this study, we screened eight cell death-inducing effectors from oomycete species in N. benthamiana and tested the requirements of ETI signaling components to induce cell death. SGT1 was required for PsAvh163- and PcRXLR25-mediated cell death, while silencing NbHSP90 abolished PcRXLR25-, PsAvh163-, PsAvh241- and PsCRN63-triggered cell death. The cell death induced by the tested effectors does not depend on EDS1, NDR1, NRG1 and ADR1. PcRXLR25- and PsAvh163-induced cell death was found to require NRC2/3/4, indicating that these two effectors are avirulence protein candidates. Finally, we found that auto-activated NRC2/3/4 also required SGT1 and HSP90 to induce hypersensitive response.

Abstract Phytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

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.

The allotetraploid Solanaceae plant Nicotiana benthamiana contains two closely related receptor-like proteins (RLPs), NbEIX2 and NbRXEG1, which regulate the recognition of VdEIX3 and PsXEG1, respectively. VdEIX3, PsXEG1, and their homologs represent two types of microbe-associated molecular patterns (MAMPs) that are widespread in diverse pathogens. Here, we report that NbRXEG1 also participates in VdEIX3 recognition. Both eix2 and rxeg1 single mutants exhibited significantly impaired but not abolished ability to mediate VdEIX3-triggered immune responses, which are nearly abolished in eix2 rxeg1 double mutants. Moreover, a dominant negative mutant of eix2 that contains a 60 bp deletion failed to respond to VdEIX3 and could suppress VdEIX3-induced cell death in the wild-type N. benthamiana. Further phylogenetic analyses showed that NbEIX2 and NbRXEG1 are obtained from different diploid ancestors by hybridization. These results demonstrate that the allotetraploid N. benthamiana recognizes two types of MAMPs by two homologous but diverged RLPs, which provides a model in which an allopolyploid plant probably exhibits defense hybrid vigor by acquiring divergent immune receptors from different ancestors.

Riptortus pedestris (Fabricius) is a polyphagous hemipteran crop pest that mainly feeds on the leguminous plants, resulting in shriveled and dimpled seeds. With recent several outbreaks in the Huang-Huai-Hai region of China, as well as in South Korea and Japan, this species has caused enormous economic losses to soybean crops. In the present study, we found that R. pedestris feeding results in local lesions at the infestation sites. To identify the key effectors that induce plant damage during feeding, the salivary glands of R. pedestris were dissected for transcriptome sequencing, and 200 putative secreted proteins were transiently expressed in N. benthamiana. Among them, three intracellular effectors (RP191, RP246, and RP302) and one apoplastic effector (RP309) were identified as necrosis-inducing proteins (NIPs), which also triggered the reactive oxidative burst. Yeast signal sequence trap and qRT-PCR analysis suggested that these proteins might be secreted into plant tissue during R. pedestris infestation. Pathogenicity assays revealed that RP191, 246, and 302 promote Phytophthora capsici infection or induce Spodoptera litura feeding by inhibiting plant immunity. RP302 is localized to the cytoplasm and nuclei, while RP191 and 246 are endoplasmic reticulum (ER) resident proteins. RP309 stimulates the expression of PTI marker genes, and its induced cell death depends on co-receptors NbBAK1 and NbSOBIR1, indicating that it is a HAMP. Bioinformatics analysis demonstrated that four NIPs are recently evolved effectors and only conserved in the Pentatomidae. In this study, saliva-secreted proteins were used as the starting point to preliminarily analyze the harm mechanism of R. pedestris, which might provide a new idea and theoretical basis for this species control.

Diseases caused by the notorious Phytophthora spp. result in enormous economic losses to crops and forests. Increasing evidence suggests that small open reading frame-encoded polypeptides (SEPs) participate in environmental responses of animals, plants, and fungi. However, it remains largely unknown whether Phytophthora pathogens produce SEPs. Here, we systematically predicted and identified 96 SEP candidates in P. capsici. Among them, three may induce stable cell death in Nicotiana benthamiana. Phytophthora-specific and conserved SEP1 facilitated P. capsici infection. PcSEP1-induced cell death is BAK1 and SOBIR1 independent and is correlated with its virulence function. Finally, PcSEP1 may be targeted to the apoplast for carrying out its functions, for which the C terminus is indispensable. Together, our results demonstrated that SEP1 is a new virulence factor, and previously unknown SEPs may act as effector proteins in Phytophthora pathogens.

Plants have responded to microbial pathogens by evolving a two-tiered immune system, involving pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). Malectin/malectin-like domain-containing receptor-like kinases (MRLKs) have been reported to participate in many biological functions in plant including immunity and resistance. However, little is known regarding the role of MRLKs in soybean immunity. This is a crucial question to address because soybean is an important source of oil and plant proteins, and its production is threatened by various pathogens. Here, we systematically identified 72 Glycine max MRLKs (GmMRLKs) and demonstrated that many of them are transcriptionally induced or suppressed in response to infection with microbial pathogens. Next, we successfully cloned 60 GmMRLKs and subsequently characterized their roles in plant immunity by transiently expressing them in Nicotiana benthamiana, a model plant widely used to study host-pathogen interactions. Specifically, we examined the effect of GmMRLKs on PTI responses and noticed that a number of GmMRLKs negatively regulated the reactive oxygen species burst induced by flg22 and chitin, and cell death triggered by XEG1 and INF1. We also analyzed the microbial effectors AvrB- and XopQ-induced hypersensitivity response and identified several GmMRLKs that suppressed ETI activation. We further showed that GmMRLKs regulate immunity probably by coupling to the immune receptor complexes. Furthermore, transient expression of several selected GmMRLKs in soybean hairy roots conferred reduced resistance to soybean pathogen Phytophthora sojae. In summary, we revealed the common and specific roles of GmMRLKs in soybean immunity and identified a number of GmMRLKs as candidate susceptible genes that may be useful for improving soybean resistance.

Anthracnose caused by plant pathogenic Colletotrichum fungi results in large economic losses in field crop production worldwide. To aid the establishment of plant host infection, Colletotrichum pathogens secrete numerous effector proteins either in apoplastic space or inside of host cells for effective colonization. Understanding these effector repertoires is critical for developing new strategies for resistance breeding and disease management. With the advance of genomics and bioinformatics tools, a large repertoire of putative effectors has been identified in Colletotrichum genomes, and the biological functions and molecular mechanisms of some studied effectors have been summarized. Here, we review recent advances in genomic identification, understanding of evolutional characteristics, transcriptional profiling, and functional characterization of Colletotrichum effectors. We also offer a perspective on future research.

Gray mold caused by Botrytis cinerea is a critical disease that results in severe postharvest losses for the apple industry. In recent years, biological control has become an increasingly effective approach for controlling postharvest diseases in fruits. Brassica plants contain abundant natural compounds with known antimicrobial activity against numerous plant pathogens. In this study, a large-scale screening of 90 mustard cultivars was conducted to evaluate their biofumigation effects against B. cinerea. Among these, one mustard cultivar named Dilong-1, displayed the highest inhibitory effect against B. cinerea, and was able to completely inhibit mycelial growth. Further investigations showed that fumigation with Dilong-1 inhibited mycelial growth, sporulation, and spore germination of B. cinerea in vitro. In addition, fumigation using Dilong-1 showed a wide antifungal spectrum, including other fruit postharvest pathogens such as Phytophthora litchii. Furthermore, apple gray mold disease severity was significantly reduced by biofumigation using Dilong-1. Importantly, fumigation with Dilong-1 did not negatively impact final apple qualities, including weight loss, firmness, and total soluble solids. These results suggested that Dilong-1 significantly inhibited gray mold decay caused by B. cinerea without affecting the quality of apple fruits. In conclusion, biological fumigation of apple fruits with the mustard cultivar Dilong-1 is a promising eco-friendly approach for controlling apple gray mold during storage and shipment.

Microbial necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) act as cytolytic toxins and immunogenic patterns in plants. Our previous work shows that cytolytic NLPs (i.e., PyolNLP5 and PyolNLP7) from the biocontrol agent Pythium oligandrum enhance plant resistance against Phytophthora pathogens by inducing the expression of plant defensins. However, the relevance between PyolNLP-induced necrosis and plant resistance activation is still unclear. Here, we find that the necrosis-inducing activity of PyolNLP5 requires amino acid residues D127 and E129 within the conserved “GHRHDLE” motif. However, PyolNLP5-mediated plant disease resistance is irrelevant to its necrosis-inducing activity and the accumulation of reactive oxygen species (ROS). Furthermore, we reveal the positive role of non-cytotoxic PyolNLPs in enhancing plant resistance against Phytophthora pathogens and the fugal pathogen Sclerotinia sclerotiorum. Similarly, non-cytotoxic PyolNLPs also activate plant defense in a cell death-independent manner and induce defensin expression. The functions of non-cytotoxic PyolNLP13/14 rely on their conserved nlp24-like peptide pattern. Synthetic Pyolnlp24s derived from both cytotoxic and non-cytotoxic PyolNLPs can induce plant defensin expression. Unlike classic nlp24, Pyolnlp24s lack the ability of inducing ROS burst in plants with the presence of Arabidopsis nlp24 receptor RLP23. Taken together, our work demonstrates that PyolNLPs enhance plant resistance in an RLP23-independent manner, which requires the conserved nlp24-like peptide pattern but is uncoupled with ROS burst and cell death.

Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity. Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology. Nevertheless, enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects. Upstream open reading frames (uORFs) are translational control elements located in the 5′ untranslated region (UTR) of eukaryotic mRNAs and may repress the translation of downstream genes. To investigate the function of three uORFs from the 5′-UTR of ACCELERATED CELL 11 (uORFsACD11), we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation. Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels. Importantly, uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves. When coupled with the PATHOGENESIS-RELATED GENE 1 (PR1) promoter, the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2 (AtLecRK-VI.2) during pathogen attack and enhance plant resistance to Phytophthora capsici. These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack, while for ensuring lower levels of protein expression at normal conditions.

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3' untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3'-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3'-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

Abstract Reactive oxygen species (ROS) signaling plays a central role in plant immune response. BPAs, referred to as binding partner 1 of accelerated cell death 11 (ACD11) (BPA1) and BPA1-like proteins, regulate ROS-mediated defense responses in Arabidopsis thaliana. However, their distribution and evolutionary characteristics in the plant lineage remain unexplored. In this study, we demonstrated that most BPA genes form a plant-specific family with expansion events observed. We found that BPA and ACD11 genes co-exist in all land plants, suggesting that this immune regulatory module may originate at the early stage of land plant emergence and contribute to their colonization. Angiosperm BPAs can be classified into four distinct groups (I-IV) in our analysis. Domain organization and motif composition are highly conserved within each group but divergent across different groups. In certain species, BPAs undergo complex alternative splicing, suggesting their regulatory and functional divergence. The protein-protein interaction network we constructed predicted additional acting partners of BPAs. The yeast two-hybrid assay revealed 15 BPA interaction pairs forming homo- or hetero-dimers. Taken together, our results provide the first synopsis of BPA evolutionary pattern and adaptations to green plant colonization.

Abstract Diseases caused by Phytophthora species seriously affect global crop production and food security. Identification of key factors involved in plant resistance is valuable for disease management. Previously, we characterized the transcriptome of Nicotiana benthamiana which was infected with Phytophthora parasitica. Here, we selected NbERF173, one of the most strongly up-regulated genes of N. benthamiana in response to P. parasitica infection, for further investigation. First, NbERF173 encodes a conserved transcription factor in our tested plant species. Second, overexpression of NbERF173 in N. benthamiana enhanced its resistance to P. parasitica, and silencing of NbERF173 significantly promoted the infection of both P. parasitica and Botrytis cinerea. Additionally, we demonstrated that NbERF173 can participate in reprograming of gene expression during P. parasitica infection and manipulate expression patterns of many defense-related genes, including two proteinase inhibitors encoding genes (PI1-B and KTI1). Furthermore, overexpression of PI1-B and KTI1 strengthened plant resistance to P. parasitica and partially restored the deficiency in resistance of NbERF173-silenced N. benthamiana plants. Finally, we found that NbERF173 could not bind to the promoters of PI1-B and KTI1 using yeast one-hybrid assay. Together, our results suggest that NbERF173 positively regulate the disease resistance, probably by reprograming of defense-related genes.

Golgi reassembly stacking proteins (GRASPs) play important roles in Golgi structure formation, ER stress response, and unconventional secretion in eukaryotic cells. However, GRASP functions in oomycetes haven’t been adequately characterized. Here, we report the identification and functional analysis of PsGRASP, a GRASP-encoding gene from the soybean-infecting oomycete Phytophthora sojae. Transcriptional profiling showed that PsGRASP expression is up-regulated at the infection stages. PsGRASP knockout mutants were created using the CRISPR/Cas9 system. These mutants exhibited impaired vegetative growth, zoospore release and virulence. PsGRASP was involved ER stress responses and altered laccase activity. Our work suggests that PsGRASP is crucial for P. sojae development and pathogenicity.

Summary: Leucine-rich repeat receptor-like kinases (LRR-RLKs) are critical signal receptors in plant development and defense. Like plants, oomycete pathogen genomes also harbor LRR-RLKs, but their functions remain largely unknown. Here, we systematically characterize all the 24 LRR-RLK genes (PsRLKs) from Phytophthora sojae, which is a model of oomycete pathogens. Although none of them was required for vegetative growth, the specific PsRLKs are important for stress responses, zoospore production, zoospores chemotaxis, and pathogenicity. Interestingly, the Gα subunit PsGPA1 interacts with the five chemotaxis-related PsRLKs via their intracellular kinase domains, and expression of PsGPA1 gene is downregulated in the three mutants (ΔPsRLK17/22/24). Moreover, we generated the PsRLK-PsRLK interaction network of P. sojae and found that PsRLK21, together with PsRLK10 or PsRLK17, regulate virulence by direct association. Taken together, our results reveal the diverse roles of LRR-RLKs in modulating P. sojae development, interaction with soybean, and responses to diverse environmental factors.

Maize seed treatment with chemicals to control underground pests is a common agricultural practice, but inappropriate use of insecticides poses a considerable threat to plant development and soil nontarget organisms. In this study, the availability of tetraniliprole seed dressing to control the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae) in the maize seeding stage and its safety to earthworms (Eisenia fetida) were investigated. The selective toxicity (ST) of tetraniliprole between E. fetida and A. ipsilon was greater than 4000. No significant adverse effect of tetraniliprole seed treatment on the germination of maize seeds was observed at concentrations of 2.4–9.6 g a.i. /kg seed. Compared with the untreated control, seed treatment with tetraniliprole at 9.6 g a.i. /kg seed greatly reduced the percentage of damaged plants from 88.73% to 26.67%, and achieved the highest control effect of 69.91%. Tetraniliprole of 2.4 g a.i. /kg seed can effectively inhibit A. ipsilon until 14 days after seed germination, with the lowest mortality rate of 44.44%. During the entire exposure period, the maximum residual concentration of tetraniliprole detected in the soil (5.86 mg/kg) was considerably lower than the LC50 value of tetraniliprole to E. fetida (>4000 mg/kg). According to the low-tier risk assessment, the highest risk quotient (RQ) of tetraniliprole seed treatment to earthworms at test concentrations was 2.8 × 10−3, which was evaluated as acceptable. This study provided data support for tetraniliprole seed treatment to control underground pests in maize fields.

Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

Crop protection strategies that are effective but that reduce our reliance on chemical pesticides are urgently needed to meet the UN sustainable development goals for global food security. Mycoparasitic oomycetes such as Pythium oligandrum and Pythium periplocum, have potential for the biological control of plant diseases that threaten crops and have attracted much attention due to their abilities to antagonize plant pathogens and modulate plant immunity. Studies of the molecular and genetic determinants of mycoparasitism in these species have been less well developed than those of their fungal counterparts. Carbohydrate-active enzymes (CAZymes) from P. oligandrum and P. periplocum are predicted to be important components of mycoparasitism, being involved in the degradation of the cell wall of their oomycete and fungal prey species. To explore the evolution of CAZymes of these species we performed an in silico identification and comparison of the full CAZyme complement (CAZyome) of the two mycoparasitic Pythium species (P. oligandrum and P. periplocum), with seven other Pythium species, and four Phytophthora species. Twenty CAZy gene families involved in the degradation of cellulose, hemicellulose, glucan, and chitin were expanded in, or unique to, mycoparasitic Pythium species and several of these genes were expressed during mycoparasitic interactions with either oomycete or fungal prey, as revealed by RNA sequencing and quantitative qRT-PCR. Genes from three of the cellulose and chitin degrading CAZy families (namely AA9, GH5_14, and GH19) were expanded via tandem duplication and predominantly located in gene sparse regions of the genome, suggesting these enzymes are putative pathogenicity factors able to undergo rapid evolution. In addition, five of the CAZy gene families were likely to have been obtained from other microbes by horizontal gene transfer events. The mycoparasitic species are able to utilize complex carbohydrates present in fungal cell walls, namely chitin and N-acetylglucosamine for growth, in contrast to their phytopathogenic counterparts. Nonetheless, a preference for the utilization of simple sugars for growth appears to be a common trait within the oomycete lineage.

Phytophthora comprises a group of filamentous plant pathogens that cause serious crop diseases worldwide. It is widely known that a complex effector repertoire was secreted by Phytophthora pathogens to manipulate plant immunity and determine resistance and susceptibility. It is also recognized that Phytophthora pathogens may inhabit natural niches within complex environmental microbes, including bacteria. However, how Phytophthora pathogens interact with their cohabited microbes remains poorly understood. Here, we present such an intriguing case by using Phytophthora–bacteria interaction as a working system. We found that under co-culture laboratory conditions, several Phytophthora pathogens appeared to block the contact of an ecologically relevant bacterium, including Pseudomonas fluorescence and a model bacterium, Escherichia coli. We further observed that Phytophthora sojae utilizes a conserved Crinkler (CRN) effector protein, PsCRN63, to impair bacterial growth. Phytophthora capsici deploys another CRN effector, PcCRN173, to interfere with bacterial flagellum- and/or type IV pilus-mediated motility whereas a P. capsici-derived RxLR effector, PcAvh540, inhibits bacterial swimming motility, but not twitching motility and biofilm formation, suggesting functional diversification of effector-mediated Phytophthora–bacteria interactions. Thus, our studies provide a first case showing that the filamentous Phytophthora pathogens could deploy effectors to interfere with bacterial growth and motility, revealing an unprecedented effector-mediated inter-kingdom interaction between Phytophthora pathogens and bacterial species and thereby uncovering ecological significance of effector proteins in filamentous plant pathogens besides their canonical roles involving pathogen–plant interaction.

Abstract Salicylic acid (SA) plays a crucial regulatory role in plant immunity. NPR1 (non-expressor of pathogenesis related-1) is a SA receptor and plays a pivotal role in SA signaling. However, pathogen effectors which target NPR1 to promote infection have rarely been reported. Here, we identified a Phytophthora capsici effector RxLR48 that associates with NPR1, facilitates P. capsici infection and is required for pathogen virulence. Furthermore, we demonstrated that RxLR48 promotes NPR1’s nuclear localization and inhibits its proteasome-mediated degradation, suggesting that RxLR48 suppresses SA signaling by targeting the central regulator NPR1. In addition, we showed that RxLR48 also suppresses pattern-triggered immunity (PTI). Together, our research indicates that P. capsici suppresses plant immunity by targeting SA and PTI pathways.

Abstract Autophagy is an evolutionarily conserved mechanism in eukaryotes with roles in development and the virulence of plant fungal pathogens. However, few reports on autophagy in oomycete species have been published. Here, we identified 26 autophagy-related genes (ATGs) belonging to 20 different groups in Phytophthora sojae using a genome-wide survey, and core ATGs in oomycetes were used to construct a preliminary autophagy pathway model. Expression profile analysis revealed that these ATGs are broadly expressed and that the majority of them significantly increase during infection stages, suggesting a central role for autophagy in virulence. Autophagy in P. sojae was detected using a GFP-PsAtg8 fusion protein and the fluorescent dye MDC during rapamycin and starvation treatment. In addition, autophagy was significantly induced during sporangium formation and cyst germination. Silencing PsAtg6a in P. sojae significantly reduced sporulation and pathogenicity. Furthermore, a PsAtg6a-silenced strain showed haustorial formation defects. These results suggested that autophagy might play essential roles in both the development and infection mechanism of P. sojae.

Valsa pyri is the causal agent of pear canker disease, which leads to enormous losses of pear production in eastern Asian, especially China. In this study, we identified a fungal-specific transcription factor 1 (termed as VpFSTF1) from V. pyri, which is highly conserved in fungi. To characterize its functions, we generated mutant and complementation strains in V. pyri and found that ΔVpFSTF1 mutants lost the ability to form fruiting bodies along with the reduced virulence. The radial growth of ΔVpFSTF1 mutant was sensitive to increasing concentrations of hydrogen peroxide (H2O2) and salicylic acid (SA). Moreover, RNA-sequencing (RNA-Seq) analysis of wild-type (WT) and ΔVpFSTF1 mutant strains was performed, and the results revealed 1,993 upregulated, and 2006 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were corresponding to the genes that are involved in amino acid metabolism, starch, and sucrose metabolism, gluconeogenesis, citrate cycle, and carbon metabolism. Interestingly, pathogen host interaction (PHI) analysis showed that 69 downregulated genes were related to virulence, suggesting that they might function downstream of VpFSTF1. Nine DEGs were further validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the results were consistent with RNA-seq analysis. Furthermore, promoter regions were predicted, and VpFSTF1 binding activity was assessed. We demonstrated that five promoters are directly or indirectly targeted by VpFSTF1, including catalase-related peroxidase (VPIG_01209) and P450 family genes. Taken together, these findings indicate that VpFSTF1 is crucial for the virulence of V. pyri via direct or indirect regulation of downstream genes expression and lay an important foundation for understanding the molecular mechanism of V. pyri infection.

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.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

As a non-pathogenic oomycete, the biocontrol agent Pythium oligandrum is able to control plant diseases through direct mycoparasite activity and boosting plant immune responses. Several P. oligandrum elicitors have been found to activate plant immunity as microbe-associated molecular patterns (MAMPs). Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are a group of MAMPs widely distributed in eukaryotic and prokaryotic plant pathogens. However, little is known about their distribution and functions in P. oligandrum and its sister species Pythium periplocum. Here, we identified a total of 25 NLPs from P. oligandrum (PyolNLPs) and P. periplocum (PypeNLPs). Meanwhile, we found that PyolNLPs/PypeNLPs genes cluster in two chromosomal segments, and our analysis suggests that they expand by duplication and share a common origin totally different from that of pathogenic oomycetes. Nine PyolNLPs/PypeNLPs induced necrosis in Nicotiana benthamiana by agroinfiltration. Eight partially purified PyolNLPs/PypeNLPs were tested for their potential biocontrol activity. PyolNLP5 and PyolNLP7 showed necrosis-inducing activity in N. benthamiana via direct protein infiltration. At sufficient concentrations, they both significantly reduced disease severity and suppressed the in planta growth of Phytophthora capsici in solanaceous plants including N. benthamiana (tobacco), Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Our assays suggest that the Phytophthora suppression effect of PyolNLP5 and PyolNLP7 is irrelevant to reactive oxygen species (ROS) accumulation. Instead, they induce the expression of antimicrobial plant defensin genes, and the induction depends on their conserved nlp24-like peptide pattern. This work demonstrates the biocontrol role of two P. oligandrum NLPs for solanaceous plants, which uncovers a novel approach of utilizing NLPs to develop bioactive formulae for oomycete pathogen control with no ROS-caused injury to plants.

Initially identified as a mammalian apoptosis suppressor, defender against apoptotic death 1 (DAD1) protein has conserved plant orthologs acting as negative regulators of cell death. The potential roles and action mechanisms of plant DADs in resistance against Phytophthora pathogens are still unknown. Here, we cloned GmDAD1 from soybean and performed functional dissection. GmDAD1 expression can be induced by Phytophthora sojae infection in both compatible and incompatible soybean varieties. By manipulating GmDAD1 expression in soybean hairy roots, we showed that GmDAD1 transcript accumulations are positively correlated with plant resistance levels against P. sojae. Heterologous expression of GmDAD1 in Nicotiana benthamiana enhanced its resistance to Phytophthora parasitica. NbDAD1 from N. benthamiana was shown to have similar role in conferring Phytophthora resistance. As an endoplasmic reticulum (ER)-localized protein, GmDAD1 was demonstrated to be involved in ER stress signaling and to affect the expression of multiple defense-related genes. Taken together, our findings reveal that GmDAD1 plays a critical role in defense against Phytophthora pathogens and might participate in the ER stress signaling pathway. The defense-associated characteristic of GmDAD1 makes it a valuable working target for breeding Phytophthora resistant soybean varieties.

Tomato yellow leaf curl virus (TYLCV) and its related begomoviruses cause fast-spreading diseases in tomato worldwide. How this virus induces diseases remains largely unclear. Here we report a noncoding RNA-mediated model to elucidate the molecular mechanisms of TYLCV-tomato interaction and disease development. The circular ssDNA genome of TYLCV contains a noncoding intergenic region (IR), which is known to mediate viral DNA replication and transcription in host cells, but has not been reported to contribute directly to viral disease development. We demonstrate that the IR is transcribed in dual orientations during plant infection and confers abnormal phenotypes in tomato independently of protein-coding regions of the viral genome. We show that the IR sequence has a 25-nt segment that is almost perfectly complementary to a long noncoding RNA (lncRNA, designated as SlLNR1) in TYLCV-susceptible tomato cultivars but not in resistant cultivars which contains a 14-nt deletion in the 25-nt region. Consequently, we show that viral small-interfering RNAs (vsRNAs) derived from the 25-nt IR sequence induces silencing of SlLNR1 in susceptible tomato plants but not resistant plants, and this SlLNR1 downregulation is associated with stunted and curled leaf phenotypes reminiscent of TYLCV symptoms. These results suggest that the lncRNA interacts with the IR-derived vsRNAs to control disease development during TYLCV infection. Consistent with its possible function in virus disease development, over-expression of SlLNR1 in tomato reduces the accumulation of TYLCV. Furthermore, gene silencing of the SlLNR1 in the tomato plants induced TYLCV-like leaf phenotypes without viral infection. Our results uncover a previously unknown interaction between vsRNAs and host lncRNA, and provide a plausible model for TYLCV-induced diseases and host antiviral immunity, which would help to develop effective strategies for the control of this important viral pathogen.

During pathogenic interactions, both the host and pathogen are exposed to conditions that induce programmed cell death (PCD). Certain aspects of PCD have been recently examined in eukaryotic microbes but not in oomycetes. Here, we identified conserved TatD proteins in Phytophthora sojae; the proteins are key components of DNA degradation in apoptosis. We selected PsTatD4 for further investigation because the enzyme is unique to the oomycete branch of the phylogenetic tree. The purified protein exhibited DNase activity in vitro. Its expression was upregulated in sporangia and later infective stages but downregulated in cysts and during early infection. Functional analysis revealed that the gene was required for sporulation and zoospore production, and the expression levels were associated with the numbers of hydrogen-peroxide-induced terminal dUTP nick end-labeling-positive cells. Furthermore, overexpression of PsTatD4 gene reduced the virulence in a susceptible soybean cultivar. Together, these data suggest that apoptosis may play different roles in the early and late infective stages of P. sojae, and that PsTatD4 is a key regulator of infection. The association of PsTatD4 and apoptosis will lay a foundation to understanding the basic biology of apoptosis and its roles in P. sojae disease cycle.

Resistance to Phytophthora sojae (Rps) genes have been widely used in soybean against root and stem rot diseases caused by this oomycete. Among 15 known soybean Rps genes, Rps1k has been the most widely used in the past four decades. Here, we show that the products of two distinct but closely linked RxLR effector genes are detected by Rps1k-containing plants, resulting in disease resistance. One of the genes is Avr1b-1, that confers avirulence in the presence of Rps1b. Three lines of evidence, including overexpression and gene silencing of Avr1b-1 in stable P. sojae transformants, as well as transient expression of this gene in soybean, indicated that Avr1b could trigger an Rps1k-mediated defense response. Some isolates of P. sojae that do not express Avr1b are nevertheless unable to infect Rps1k plants. In those isolates, we identified a second RxLR effector gene (designated Avr1k), located 5 kb away from Avr1b-1. Silencing or overexpression of Avr1k in P. sojae stable transformants resulted in the loss or gain, respectively, of the avirulence phenotype in the presence of Rps1k. Only isolates of P. sojae with mutant alleles of both Avr1b-1 and Avr1k could evade perception by the soybean plants carrying Rps1k.