PAMP-triggered Immunity Research Articles

Bursaphelenchus xylophilus Venom Allergen-Like Protein BxVAP1, Triggering Plant Defense-Related Programmed Cell Death, Plays an Important Role in Regulating Pinus massoniana Terpene Defense Responses.

Bursaphelenchus xylophilus (pine wood nematode, PWN), a migratory plant-parasitic nematode, acts as an etiological agent, inflicting considerable damage to pine forests worldwide. Plant immunity constitutes a crucial factor in resisting various pathogenic invasions. The primary defensive responses of host pines against PWN infection encompass terpene accumulation, defense response-related gene expression, and programmed cell death. Venom allergen-like proteins (VAPs), as potential effectors, are instrumental in facilitating the successful colonization of PWNs. In this study, we investigated the inhibition of B. xylophilus VAP (BxVAP1) expression by RNA interference in vitro. Following BxVAP1 silencing, the reproduction rate and migration rate of the PWN population in Pinus massoniana decreased, the expression of the α-pinene synthase gene was induced, other terpene synthase and pathogenesis-related genes were inhibited and delayed, the peak times and levels of terpene-related substances were changed, and the degree of cavitation in P. massoniana was diminished. Transient expression of BxVAP1 in Nicotiana benthamiana revealed that BxVAP1 was expressed in both the cell membrane and nucleus, inducing programmed cell death and the expression of pathogen-associated molecular pattern-triggered immunity marker genes (NbAcre31 and NbPTI5). This study is the first to demonstrate that silencing the BxVAP1 gene affects host defense responses, including terpenoid metabolism in P. massoniana, and that BxVAP1 can be recognized by N. benthamiana as an effector to trigger its innate immunity, expanding our understanding of the parasitic mechanism of B. xylophilus.

Overexpression of the receptor-like cytoplasmic kinase SZE1 positively regulates the PTI signaling pathway in Arabidopsis

Plants resist pathogen attacks through innate immune responses. RLCK (Receptor-like cytoplasmic kinase) is essential for plant innate immunity, but only a few RLCK functions have been discovered. SZE1 (Suppressor of ZED1-D1) was reported to be involved in the ETI (Effector-triggered Immunity) signaling pathway, but by analyzing the transcriptome data, we found that SZE1 was upregulated under flg22 (22 amino acids at flagellin's N-terminus) and Pseudomonas syringae treatment. The function of the SZE1 gene in the PTI signaling pathway was studied using SZE1 overexpression and mutant plants. While overexpression of the SZE1 gene enhanced PTI (PAMP-triggered immunity) signal transduction and the ability of Arabidopsis to resist P.s.t. DC3000 (Pseudomonas syringae pv tomato DC3000), sze1 mutants were more sensitive to pathogen infection. We also found that SZE1 could interact with BAK1 (BRI1-associated receptor kinase 1) by yeast two-hybrid and Co-IP (Co-immunoprecipitation) assays. Our results demonstrate that SZE1 positively regulates the PTI signaling pathway, which may enhance flg22-triggered PTI signal transduction by interacting with BAK1 to form a complex. Although PTI and ETI immune responses involve different activation mechanisms, eventually, they converge into many similar downstream responses. Our results provide evidence that SZE1 is a signaling element that participates in both PTI and ETI.

A Mitogen-Activated Protein Kinase Pathway Is Required for Bacillus amyloliquefaciens PMB05 to Enhance Disease Resistance to Bacterial Soft Rot in Arabidopsis thaliana.

When a plant is infected by a pathogen, endogenous immune responses are initiated. When the initiation of these defense responses is induced by a pathogen-associated molecular pattern (PAMP) of a pathogen, it is called PAMP-triggered immunity (PTI). Previous studies have shown that Bacillus amyloliquefaciens PMB05 can enhance PTI signals and improve disease control of bacterial soft rot and wilt in Arabidopsis thaliana. In the context of controlling bacterial wilt disease, the involvement of a mitogen-activated protein kinase (MAPK) signaling pathway has been established. Nevertheless, it remains unclear whether this pathway is also required for B. amyloliquefaciens PMB05 in controlling bacterial soft rot. In this study, A. thaliana ecotype Columbia (Col-0) and its mutants on a MAPK pathway-related pathway were used as a model and established that the ability of B. amyloliquefaciens PMB05 to control soft rot requires the participation of the MAPK pathway. Moreover, the enhancement of disease resistance by PMB05 is highly correlated with the activation of reactive oxygen species generation and stomata closure, rather than callose deposition. The spray inoculation method was used to illustrate that PMB05 can enhance stomatal closure, thereby restricting invasion by the soft rot bacterium. This control mechanism has also been demonstrated to require the activation of the MAPK pathway. This study demonstrates that B. amyloliquefaciens PMB05 can accelerate stomata closure via the activation of the MAPK pathway during PTI, thereby reducing pathogen invasion and achieving disease resistance against bacterial soft rot.

A Rapid Method for Screening Pathogen-Associated Molecular Pattern-Triggered Immunity-Intensifying Microbes.

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.

TaRLK2.4, a transgressive expression receptor like kinase, improves powdery mildew resistance in wheat

Plants form two immune systems, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI), to combat Blumeria graminis f. sp. tritici (Bgt) infection during the evolutionary process. In PTI, receptor-like kinases (RLKs) play important roles during pathogen infections. Based on our previous reports, there were 280 TaRLKs identified in early response to powdery mildew infection, which were divided into 34 subfamilies in this study. Differences in gene structures, cis-acting elements, and expression levels implied the function diversity of TaRLKs. TaRLK2.4, a member of LRK10L-RLKs subfamily, contained 665 amino acids, and located on the cell membrane. The main objective of this study was to investigate the role of the receptor-like kinase gene TaRLK2.4 in conferring powdery mildew resistance in wheat. Real-time quantitative PCR results indicated that TaRLK2.4 expressed during Bgt infection process, and exhibited a transgressive expression characteristic in disease resistance NILs (BJ-1). To elucidate the function of TaRLK2.4 during Bgt infection, the comprehensive analysis of virus induced gene silence and over-expression demonstrated that TaRLK2.4 promoted powdery mildew resistance positively. In summary, these results contribute to a deeper understanding of the complex and diverse biological functions of RLKs, and provide new genetic resources for wheat molecular breeding.

Interactive regulation of immune-related resistance genes with salicylic acid and jasmonic acid signaling in systemic acquired resistance in the Xanthomonas–Brassica pathosystem

Pathogen-responsive immune-related genes (resistance genes [R-genes]) and hormones are crucial mediators of systemic acquired resistance (SAR). However, their integrated functions in regulating SAR signaling components in local and distal leaves remain largely unknown. To characterize SAR in the Xanthomonas campestris pv. campestris (Xcc)–Brassica napus pathosystem, the responses of R-genes, (leaf and phloem) hormone levels, H2O2 levels, and Ca2+ signaling-related genes were assessed in local and distal leaves of plants exposed to four Xcc-treatments: Non-inoculation (control), only secondary Xcc-inoculation in distal leaves (C-Xcc), only primary Xcc-inoculation in local leaves (Xcc), and both primary and secondary Xcc-inoculation (X-Xcc). The primary Xcc-inoculation provoked disease symptoms as evidenced by enlarged destructive necrosis in the local leaves of Xcc and X-Xcc plants 7 days post-inoculation. Comparing visual symptoms in distal leaves 5 days post-secondary inoculation, yellowish necrotic lesions were clearly observed in non Xcc-primed plants (C-Xcc), whereas no visual symptom was developed in Xcc-primed plants (X-Xcc), demonstrating SAR. Pathogen resistance in X-Xcc plants was characterized by distinct upregulations in expression of the PAMP-triggered immunity (PTI)-related kinase-encoding gene, BIK1, the (CC-NB-LRR-type) R-gene, ZAR1, and its signaling-related gene, NDR1, with a concurrent enhancement of the kinase-encoding gene, MAPK6, and a depression of the (TIR-NB-LRR-type) R-gene, TAO1, and its signaling-related gene, SGT1, in distal leaves. Further, in X-Xcc plants, higher salicylic acid (SA) and jasmonic acid (JA) levels, both in phloem and distal leaves, were accompanied by enhanced expressions of the SA-signaling gene, NPR3, the JA-signaling genes, LOX2 and PDF1.2, and the Ca2+-signaling genes, CAS and CBP60g. However, in distal leaves of C-Xcc plants, an increase in SA level resulted in an antagonistic depression of JA, which enhanced only SA-dependent signaling, EDS1 and NPR1. These results demonstrate that primary Xcc-inoculation in local leaves induces resistance to subsequent pathogen attack by upregulating BIK1-ZAR1-mediated synergistic interactions with SA and JA signaling as a crucial component of SAR.

Insights into bs5 resistance mechanisms in pepper against Xanthomonas euvesicatoria through transcriptome profiling

BackgroundBacterial spot of pepper (BSP), caused by four different Xanthomonas species, primarily X. euvesicatoria (Xe), poses a significant challenge in pepper cultivation. Host resistance is considered the most important approach for BSP control, offering long-term protection and sustainability. While breeding for resistance to BSP for many years focused on dominant R genes, introgression of recessive resistance has been a more recent focus of breeding programs. The molecular interactions underlying recessive resistance remain poorly understood.ResultsIn this study, transcriptomic analyses were performed to elucidate defense responses triggered by Xe race P6 infection by two distinct pepper lines: the Xe-resistant line ECW50R containing bs5, a recessive resistance gene that confers resistance to all pepper Xe races, and the Xe-susceptible line ECW. The results revealed a total of 3357 upregulated and 4091 downregulated genes at 0, 1, 2, and 4 days post-inoculation (dpi), with the highest number of differentially expressed genes (DEGs) observed at 2 dpi. Pathway analysis highlighted DEGs in key pathways such as plant-pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, and photosynthesis – antenna proteins, along with cysteine and methionine metabolism. Notably, upregulation of genes associated with PAMP-Triggered Immunity (PTI) was observed, including components like FLS2, Ca-dependent pathways, Rboh, and reactive oxygen species (ROS) generation. In support of these results, infiltration of ECW50R leaves with bacterial suspension of Xe led to observable hydrogen peroxide accumulation without a rapid increase in electrolyte leakage, suggestive of the absence of Effector-Triggered Immunity (ETI). Furthermore, the study confirmed that bs5 does not disrupt the effector delivery system, as evidenced by incompatible interactions between avirulence genes and their corresponding dominant resistant genes in the bs5 background.ConclusionOverall, these findings provide insights into the molecular mechanisms underlying bs5-mediated resistance in pepper against Xe and suggest a robust defense mechanism in ECW50R, primarily mediated through PTI. Given that bs5 provides early strong response for resistance, combining this resistance with other dominant resistance genes will enhance the durability of resistance to BSP.

Green Routes: Exploring Protein-Based Virus-like Nanoparticle Transport and Immune Activation in Nicotiana benthamiana for Biotechnological Applications.

Viral, bacterial, fungal, and nematode infections cause significant agricultural losses, with limited treatment options, necessitating novel approaches to enhance plant defense systems and protection against pathogens. Virus-like nanoparticles (VLPs), extensively used in animal and human therapies (e.g., vaccines and immune enhancers), hold potential for novel agricultural solutions and advancing plant nanotechnology. This study employed various methodologies, including VLP production, confocal microscopy, and real-time qPCR. Our findings demonstrated the presence of 30 nm Qβ-VLPs, fluorescently labeled, within the intercellular space of Nicotiana benthamiana leaves one hour post-infiltration. Furthermore, infiltration with Qβ-VLPs led to an upregulation of key defense genes (NbPR1a, NbPR5, NbNPR, NbERF1, NbMYC2, and NbLRR2) in treated plants. Using RT-qPCR, a significant increase in the relative expression levels of defense genes was observed, with sustained high levels of NbERF1 and NbLRR2 even after 24 h. These findings suggest that Qβ-VLPs effectively upregulate genes crucial for pathogen defense in N. benthamiana, initiating PAMP-triggered immunity and launching signaling cascades that enhance defense mechanisms. This innovative application of VLPs to activate plant defense programs advances plant nanobiotechnology, offering new agricultural solutions.

Multiple Chitin- or Avirulent Strain-Triggered Immunity Induces Microbiome Reassembly in Rice.

Magnaporthe oryzae is one of the most important fungal pathogens of rice. Chitin and avirulent strains can induce two layers of immunity response, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI), in rice with cognate R genes. However, little is known about the assembly of the rice microbiome induced by PTI and ETI in rice. In this study, we investigate the impact of continuous treatment of the avirulent M. oryzae strain with AvrPi9 and chitin on the bacterial endophytic community of rice varieties harboring resistant gene Pi9 and their antagonistic activity against rice blast fungus. Analysis of the 16S rRNA showed a significant increase in the diversity and microbial co-occurrence network complexity and the number of beneficial taxa-Bacillus, Pseudomonas, Microbacterium, and Stenotrophomonas spp.-following the chitin and avirulent strain treatments. The antifungal assay with bacterial endophytes recovered from the leaves showed few bacteria with antagonistic potential in rice treated with avirulent strains, suggesting that the sequential treatment of the avirulent strain decreased the antagonistic bacteria against M. oryzae. Moreover, we identified Bacillus safensis Ch_66 and Bacillus altitudinis Nc_68 with overall antagonistic activities in vivo and in vitro. Our findings provide a novel insight into rice microbiome assembly in response to different innate immunity reactions.

The soybean plasma membrane GmDR1 protein conferring broad-spectrum disease and pest resistance regulates several receptor kinases and NLR proteins

Overexpression of Glycine max disease resistant 1 (GmDR1) exhibits broad-spectrum resistance against Fusarium virguliforme, Heterodera glycines (soybean cyst nematode), Tetranychus urticae (Koch) (spider mites), and Aphis glycines Matsumura (soybean aphids) in soybean. To understand the mechanisms of broad-spectrum immunity mediated by GmDR1, the transcriptomes of a strong and a weak GmDR1-overexpressor following treatment with chitin, a pathogen- and pest-associated molecular pattern (PAMP) common to these organisms, were investigated. The strong and weak GmDR1-overexpressors exhibited altered expression of 6098 and 992 genes, respectively, as compared to the nontransgenic control following chitin treatment. However, only 192 chitin- and 115 buffer-responsive genes exhibited over two-fold changes in expression levels in both strong and weak GmDR1-overexpressors as compared to the control. MapMan analysis of the 192 chitin-responsive genes revealed 64 biotic stress-related genes, of which 53 were induced and 11 repressed as compared to the control. The 53 chitin-induced genes include nine genes that encode receptor kinases, 13 encode nucleotide-binding leucine-rich repeat (NLR) receptor proteins, seven encode WRKY transcription factors, four ethylene response factors, and three MYB-like transcription factors. Investigation of a subset of these genes revealed three receptor protein kinases, seven NLR proteins, and one WRKY transcription factor genes that are induced following F. virguliforme and H. glycines infection. The integral plasma membrane GmDR1 protein most likely recognizes PAMPs including chitin and activates transcription of genes encoding receptor kinases, NLR proteins and defense-related genes. GmDR1 could be a pattern recognition receptor that regulates the expression of several NLRs for expression of PAMP-triggered immunity and/or priming the effector triggered immunity.

Unraveling Microbial Strategies: Targeting the Plant Ubiquitin - Proteasome Pathway

The Ubiquitin-Proteasome System (UPS) stands as a central regulator in the intricate web of plant immune responses, orchestrating the turnover of key immune components. Through the UPS, plants finely tune their defenses, from the initial recognition of pathogens to the activation of defense strategies. Immune receptors, signaling components associated with defense hormones, and transcription factors are among the targets modulated by the UPS, shaping both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). However, pathogens have evolved sophisticated strategies to manipulate the UPS for their benefit. Fungi, bacteria, and viruses deploy effector proteins to exploit the UPS, either by promoting the degradation of host defense elements or by interfering with UPS-mediated processes. This intricate interplay underscores the pivotal role of the UPS in plant-pathogen dynamics, presenting both challenges and opportunities for understanding and manipulating plant immunity. Unraveling the complexities of UPS regulation and pathogen exploitation thereof is crucial for advancing our knowledge of plant-microbe interactions and developing strategies for sustainable disease management in agriculture.

An effector SsCVNH promotes the virulence of Sclerotinia sclerotiorum through targeting class III peroxidase AtPRX71.

Many plant pathogens secrete effector proteins into the host plant to suppress host immunity and facilitate pathogen colonization. The necrotrophic pathogen Sclerotinia sclerotiorum causes severe plant diseases and results in enormous economic losses, in which secreted proteins play a crucial role. SsCVNH was previously reported as a secreted protein, and its expression is significantly upregulated at 3 h after inoculation on the host plant. Here, we further demonstrated that deletion of SsCVNH leads to attenuated virulence. Heterologous expression of SsCVNH in Arabidopsis enhanced pathogen infection, inhibited the host PAMP-triggered immunity (PTI) response and increased plant susceptibility to S. sclerotiorum. SsCVNH interacted with class III peroxidase AtPRX71, a positive regulator of innate immunity against plant pathogens. SsCVNH could also interact with other class III peroxidases, thus reducing peroxidase activity and suppressing plant immunity. Our results reveal a new infection strategy employed by S. sclerotiorum in which the fungus suppresses the function of class III peroxidases, the major component of PTI to promote its own infection.

Phylogenetic Analysis of Wall-Associated Kinase Genes in Triticum Species and Characterization of TaWAK7 Involved in Wheat Powdery Mildew Resistance.

Wall-associated kinases (WAKs), a group of receptor-like kinases, have been found to play important roles in defending against pathogens and in various developmental processes. However, the importance of this family in wheat remains largely unknown. Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), which initiates infection on the cell surface and forms haustoria inside the cell; therefore, the defense to Bgt involves extracellular and subsequently intracellular signals. In this study, WAKs were identified genome-wide and analyzed phylogenetically, and then a transmembrane WAK gene that putatively participated in pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity to Bgt was functionally and evolutionarily investigated. In total, 1,193 WAKs were identified from wheat and its Gramineae relatives. Phylogenetic analysis indicated that WAKs expanded through tandem duplication or segment duplication. TaWAK7, from chromosome 2A, was identified as a Bgt-inducible gene both in susceptible and resistant materials, but it showed distinct responsive patterns. Functional analysis showed that TaWAK7 was involved in both the basal and resistance gene-mediated resistances. The specific gene structures and protein characteristics of TaWAK7, along with its orthologs, were characterized both in subgenomes of Triticum spp. and in the A genome of multiple wheat accessions, which revealed that TaWAK7 orthologs underwent complex evolution with frequent gene fusion and domain deletion. In addition, three cytoplasmic proteins interacting with TaWAK7 were indicated by yeast two-hybrid and bimolecular fluorescence complementation assays. Binding of TaWAK7 with these proteins could change its subcellular localization from the plasma membrane to the cytoplasm. This study provides a better understanding of the evolution of WAKs at the genomic level and TaWAK7 at the gene level and provides useful clues for further investigation of how WAKs transmit the extracellular signals to the cytoplasm to activate defense responses.

Comparative transcriptomic profiling of the two-stage response of rice to Xanthomonas oryzae pv. oryzicola interaction with two different pathogenic strains.

Two-tiered plant immune responses involve cross-talk among defense-responsive (DR) genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI) and effector-triggered susceptibility (ETS). Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important bacterial disease that causes serious threats to rice yield and quality. Transcriptomic profiling provides an effective approach for the comprehensive and large-scale detection of DR genes that participate in the interactions between rice and Xoc. In this study, we used RNA-seq to analyze the differentially expressed genes (DEGs) in susceptible rice after inoculation with two naturally pathogenic Xoc strains, a hypervirulent strain, HGA4, and a relatively hypovirulent strain, RS105. First, bacterial growth curve and biomass quantification revealed that differential growth occurred beginning at 1day post inoculation (dpi) and became more significant at 3 dpi. Additionally, we analyzed the DEGs at 12h and 3 days post inoculation with two strains, representing the DR genes involved in the PTI and ETI/ETS responses, respectively. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the common DEGs, which included 4380 upregulated and 4019 downregulated genes and 930 upregulated and 1383 downregulated genes identified for the two strains at 12h post inoculation (hpi) and 3 dpi, respectively. Compared to those at 12 hpi, at 3 dpi the number of common DEGs decreased, while the degree of differential expression was intensified. In addition, more disease-related GO pathways were enriched, and more transcription activator-like effector (TALE) putative target genes were upregulated in plants inoculated with HGA4 than in those inoculated with RS105 at 3 dpi. Then, four DRs were randomly selected for the BLS resistance assay. We found that CDP3.10, LOC_Os11g03820, and OsDSR2 positively regulated rice resistance to Xoc, while OsSPX3 negatively regulated rice resistance. By using an enrichment method for RNA-seq, we identified a group of DEGs related to the two stages of response to the Xoc strain, which included four functionally identified DR genes.

Molecular Mechanisms and Cytopathology of Phytophthora: Strategies, Interactions and Future Perspectives

Phytophthora is an aggressive plant pathogen, pose substantial threats to global agriculture, leading to extensive crop losses. Controlling Phytophthora diseases remains challenging, with limited effective methods available. Host resistance emerges as a promising strategy, but its sustainability hinges on a profound comprehension of the intricate molecular dynamics governing Phytophthora-plant interactions. These interactions unveil a hemi-biotrophic lifestyle of Phytophthora, transitioning from biotrophic to necrotrophic phases during infection. The infection process involves a series of orchestrated events, including chemotactic attraction, penetration, and sporulation on the host surface. Molecular cytology elucidates a sophisticated interplay between Phytophthora and plant defense mechanisms. Phytophthora elicits defense responses in host plants through the release of elicitor molecules, triggering PAMP Triggered Immunity (PTI) responses such as antimicrobial compound production and cell wall fortification. Detection and recognition of Phytophthora effectors instigate a second layer of defense in resistant plants, leading to Effector Triggered Immunity (ETI) and hypersensitive response. In response, virulent strains evolve altered effectors to evade detection and suppress host defenses, underscoring the ongoing molecular coevolution between Phytophthora and plants. The pathogenic success of Phytophthora species is attributed to their diverse and rapidly evolving effector gene complements, targeting various host proteins and cellular processes. Future strategies for combating Phytophthora diseases include genome editing using CRISPR/Cas-9 technology to enhance plant immunity and the identification of non-race-specific resistance sources for broad-spectrum protection. In essence, a comprehensive understanding of Phytophthora-plant interactions at the molecular level is imperative for devising effective strategies to mitigate their impact on global agriculture.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Class II diacylglycerol kinases participate in the basal immune responses of Nicotiana benthamiana to Ralstonia solanacearum

Phospholipid signaling plays an important role in Nicotiana benthamiana immune responses to phytopathogenic bacteria. In this study, we isolated three orthologs encoding class II diacylglycerol kinase (NbDGK4-1, NbDGK4-2 and NbDGK7) in the N. benthamiana genome. In contrast to the undetectable expression of NbDGK4-2, the NbDGK4-1 and NbDGK7 expression levels increased in response to Ralstonia solanacearum and the flg22 peptide. The induction of the hypersensitive response was not affected, but bacterial growth increased and the onset of bacterial wilt symptoms was accelerated in the NbDGK4-1-silenced and NbDGK7-silenced plants challenged with R. solanacearum. The expression levels of NbPR-1 and NbPR-4 (marker genes for salicylic acid and jasmonic acid signaling, respectively) decreased in the NbDGK4-1-silenced and NbDGK7-silenced plants inoculated with R. solanacearum. The expression of pathogen-associated molecular pattern-triggered immunity (PTI) marker genes were also inhibited in the NbDGK4-1-silenced and NbDGK7-silenced plants infiltrated with the type III secretion system (T3SS)-deficient R. solanacearum mutant or flg22. Accordingly, the T3SS-deficient R. solanacearum bacterial population increased in the NbDGK4-1-silenced and NbDGK7-silenced plants. Moreover, flg22-induced resistance and callose deposition were also compromised in the NbDGK4-1-silenced and NbDGK7-silenced plants. Considered together, the study results indicate class II diacylglycerol kinases may be important for N. benthamiana basal disease resistance (e.g., PTI responses).

Unraveling the role of microRNA-like RNAs in enhancing Puccinia triticina pathogenicity in wheat by high-throughput sequencing data

Wheat (Triticum aestivum L.), a staple crop cultivated across an estimated 215 million hectares worldwide, encounters significant yield reductions due to the leaf rust disease, instigated by the fungus Puccinia triticina (Pt). Control strategies for this pathogen are limited. Research on wheat-leaf rust interaction in the past few years has mainly focused on characterizing and modulating the performance of resistance (R) and other defense genes of the host plant, but rarely targeted to decipher the network of molecular strategies employed by the pathogen except secretome analysis. In wheat plants, pathogen may induce a greater diversity of small RNAs (sRNAs) to manipulate the host's gene expression and suppress defense responses. In the present study, Pt microRNA-like-RNAs (milRNAs) were screened and identified from sRNA libraries prepared from leaf rust pathogen-infected leaves of wheat near isogenic lines (NILs). Pathogenesis engendering Pt target genes were validated through degradome libraries. Our study indicates that milRNAs enter the host system to suppress its immunity and promote symbiosis-like association to maintain a longer virulence span. Through RT‒qPCR expression analysis, it was suggested that milRNAs create an environment to enhance symbiosis via cross-kingdom silencing of host disease resistance gene encoding proteins. We found that seven out of eight milRNAs effectively regulate pathogenesis-related genes and repressed the disease-resistance genes in host wheat. Functional annotation of target genes of Pt reveals regulation of vesicle synthesis, transport, and fusion, which represents cross-kingdom vesicle-mediated export of milRNAs. Our results also manifest an integrative view of how virulence-associated milRNAs endeavor to stamp out PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) to increase susceptibility of the host. This study epitomizes one of the tactics developed by Pt employing milRNAs in regulating virulence during leaf rust infection and will usher to design strategies to manage pathogenesis to protect wheat immunity against leaf rust disease.

Fusarium graminearum rapid alkalinization factor peptide negatively regulates plant immunity and cell growth via the FERONIA receptor kinase.

The plant rapid alkalinization factor (RALF) peptides function as key regulators in cell growth and immune responses through the receptor kinase FERONIA (FER). In this study, we report that the transcription factor FgPacC binds directly to the promoter of FgRALF gene, which encodes a functional homologue of the plant RALF peptides from the wheat head blight fungus Fusarium graminearum (FgRALF). More importantly, FgPacC promotes fungal infection via host immune suppression by activating the expression of FgRALF. The FgRALF peptide also exhibited typical activities of plant RALF functions, such as inducing plant alkalinization and inhibiting cell growth, including wheat (Triticum aestivum), tomato (Solanum lycopersicum) and Arabidopsis thaliana. We further identified the wheat receptor kinase FERONIA (TaFER), which is capable of restoring the defects of the A. thaliana FER mutant. In addition, we found that FgRALF peptide binds to the extracellular malectin-like domain (ECD) of TaFER (TaFERECD) to suppress the PAMP-triggered immunity (PTI) and cell growth. Overexpression of TaFERECD in A. thaliana confers plant resistance to F. graminearum and protects from FgRALF-induced cell growth inhibition. Collectively, our results demonstrate that the fungal pathogen-secreted RALF mimic suppresses host immunity and inhibits cell growth via plant FER receptor. This establishes a novel pathway for the development of disease-resistant crops in the future without compromising their yield potential.

DGS1 improves rice disease resistance by elevating pathogen-associated molecular pattern-triggered immunity.

The online version contains supplementary material available at 10.1007/s42994-024-00137-9.