The receptor-like cytoplasmic kinase RIPK regulates broad-spectrum ROS signaling in multiple layers of plant immune system

Production of reactive oxygen species(ROS)via the activity of respiratory burst oxidase homologs(RBOHs)plays a vital role in multiple layers of the plant immune system,including pathogen-associated molecular pattern-triggered immunity(PTI),damage-associated molecular pattern-triggered immunity(DTI),effector-triggered immunity(ETI),and systemic acquired resistance(SAR).It is generally established that RBOHD is activated by different receptor-like cytoplasmic kinases(RLCKs)in response to various immune elicitors.In this study,we showed that RPM1-INDUCED PROTEIN KINASE(RIPK),an RLCK VII subfamily member,contributes to ROS production in multiple layers of plant immune system.The ripk mutants showed reduced ROS production in response to treatment with all examined immune elicitors that trigger PTI,DTI,ETI,and SAR.We found that RIPK can directly phosphorylate the N-terminal region of RBOHD in vitro,and the levels of phosphorylated S343/S347 residues of RBOHD are sigfniciantly lower in ripk mutants compared with the wild type upon treatment with all tested immune elicitors.We further demonstrated that phosphorylation of RIPK is required for its function in regulating RBOHD-mediated ROS production.Similar to rbohd,ripk mutants showed reduced stomatal closure and impaired SAR,and were susceptible to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our results indicate that RIPK regulates broad-spectrum RBOHD-mediated ROS signaling during PTI,DTI,ETI,and SAR,leading to subsequent RBOHD-dependent immune responses.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops.Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear.A glycine-rich nuclear effector,VdCE51,was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum.This effector was a required factor for full virulence of V.dahliae,and its nuclear localization was a requisite for suppressing plant immunity.The thioredoxin GhTRXH2,identified as a positive regulator of plant immunity,was a host target of VdCE51.Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes,and the SA signaling pathway by inhibiting the accumulation of GhTRXH2,thus suppressing plant immunity.

A novel chorismate mutase effector secreted from root-knot nematode Meloidogyne enterolobii manipulates plant immunity to promote parasitism

Meloidogyne spp.is an economically important plant-parasitic nematode distributed worldwide.To fight with host immune system for successful parasitism,plant parasitic nematodes secrete effectors to promote infection.In this study,we identified one chorismate mutase(CM)effector from M.enterolobii,named Me-CM.Spatial and temporal expression assays exhibited Me-cm is expressed in esophageal glands and up-regulated at parasitic-stage juveniles.Me-CM affects the pathogenicity of M.enterolobii based on the reduced infection rate,number of galls,egg masses,eggs per mass and multiplication rate collected from RNA silencing experiments.We showed that Me-CM localized in the cytoplasm and nucleus of plant cells and decreased the expression level of the marker gene PR1 of salicylic acid(SA)pathway.Besides,constitutive expression of Me-cm in Arabidopsis thaliana significantly reduced salicylic acid concentration.These results suggested that M.enterolobii may secrete effector Me-CM to fight with plantimmunesystemsvia regulating SA signaling pathway when interacting with host plants,ultimately facilitating parasitism.

The IDD Transcription Factors:Their Functions in Plant Development and Environmental Response

INDETERMINATE-DOMAIN proteins(IDDs)are a plant-specific transcription factor family characterized by a conserved ID domain with four zinc finger motifs.Previous studies have demonstrated that IDDs coordinate a diversity of physiological processes and functions in plant growth and development,including floral transition,plant architecture,seed and root development,and hormone signaling.In this review,we especially summarized the latest knowledge on the functions and working models of IDD members in Arabidopsis,rice,and maize,particularly focusing on their role in the regulatory network of biotic and abiotic environmental responses,such as gravity,temperature,water,and pathogens.Understanding these mechanisms underlying the function of IDD proteins in these processes is important for improving crop yields by manipulating their activity.Overall,the review offers valuable insights into the functions and mechanisms of IDD proteins in plants,providing a foundation for further research and potential applications in agriculture.

Popularization and Application of Haidaosu(5% Amino-oligosaccharin)as a Plant Immunity Inducer in Main Crops Cultured by Corps

The plant immunity inducer, amino-oligosaccharin, has remarkable effects in disease resistance, cold tolerance, growth promotion, yield increase and quality improvement. This paper introduced the action mechanism of amino-oligosaccharin, its main application effects on crops and application techniques. In 2013-2014, ex- periments were conducted on a variety of crops at multiple locations by Xinjiang Corps as well as popularization and application in 2013-2014, and it was shown by the popularization and application that the application of amino-oligosaccharin could promote plant growth, reduce the incidence of crop diseases and improve crop yield and product quality.

ORYZA SATIVA SPOTTED-LEAF 41(OsSPL41) Negatively Regulates Plant Immunity in Rice

Identification of immunity-associated leucine-rich repeat receptor-like protein kinases(LRR-RLK) is critical to elucidate the LRR-RLK mediated mechanism of plant immunity.Here,we reported the map-based cloning of a novel rice SPOTTED-LEAF 41(Os SPL41) encoding a putative LRR-RLK protein(Os LRR-RLK41/Os SPL41) that regulated disease responses to the bacterial blight pathogen Xanthomonas oryzae pv.oryzae(Xoo).An 8-bp insertion at position 865 bp in a mutant spotted-leaf 41(spl41) allele led to the formation of purple-brown lesions on leaves.Functional complementation by the wild type allele(Os SPL41) can rescue the mutant phenotype,and the complementary lines showed similar performance to wild type in a number of agronomic,physiological and molecular indices.Os SPL41 was constitutively expressed in all tissues tested,and Os SPL41 contains a typical transmembrane domain critical for its localization to the cell membrane.The mutant exhibited an enhanced level of resistance to Xoo in companion of markedly up-regulated expression of pathogenesis-related genes such as Os PR10a,Os PAL1 and Os NPR1,while the level of salicylic acid was significantly increased in spl41.In contrast,the over-expression lines exhibited a reduced level of H_(2)O_(2) and were much susceptible to Xoo with down-regulated expression of pathogenesis-related genes.These results suggested that Os SPL41 might negatively regulate plant immunity through the salicylic acid signaling pathway in rice.

Recent advances in plant immunity with cell death:A review

Cell death is an important physiological phenomenon in life.It can be programmed or unprogrammed.Unprogrammed cell death is usually induced by abiotic or biotic stress.Recent studies have shown that many proteins regulate both cell death and immunity in plants.Here,we provide a review on the advances in plant immunity with cell death,especially the molecular regulation and underlying mechanisms of those proteins involved in both cell death and plant immunity.In addition,we discuss potential approaches toward improving plant immunity without compromising plant growth.

A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Egg-associated secretions from the brown planthopper(Nilaparvata lugens)activate rice immune responses

The brown planthopper(BPH,Nilaparvata lugens)is a notorious sap-sucking insect pest that damages rice(Oryza sativa)plants throughout Asia.During BPH feeding,saliva enters rice plant tissues,whereas during oviposition egg-associated secretions(EAS)are deposited in damaged plant tissue.Dynamic changes in rice to planthopper salivary effectors have been widely reported.However,the effects of EAS from planthopper on rice immunity remains largely unexplored.In this study,we found that both infestation of rice by gravid BPH female adults and treatment with the EAS elicited a strong and rapid accumulation of jasmonic acid(JA),JA-isoleucine,and hydrogen peroxide in rice.EAS enhanced plant defenses not only in rice but also in tobacco,and these impaired the performance of BPH on rice,as well as the performance of aphids and whiteflies on tobacco.High-throughput proteome sequencing of EAS led to 110 proteins being identified and 53 proteins with 2 or more unique peptides being detected.Some proteins from BPH EAS were also found in the salivary proteome from herbivores,suggesting potential evolutionary conservation of effector functions across feeding and oviposition;however,others were only identified in EAS,and these are likely specifically related to oviposition.These findings point to novel proteins affecting interactions between planthoppers and rice during oviposition,providing an additional source of information for effector studies.

Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

A novel ABA structural analogues enhanced plant resistance by inducing the plant immunity and inactivating ABA signaling pathway

Abscisic acid(ABA)is a phytohormone that not only important for plant growth,but also mediating the stress response.The roles of ABA in plant immunity are especially multifaceted.Recently,the ABA functional analogues are of great significance to promote its application.Here,we reported an ABA functional analogue named 167A.167A inhibits plant growth and seeds germinating of Arabidopsis.Meanwhile,the 167A enhanced the plant immunity,which is opposite of ABA.We further investigated the PTI-response after 167A treatment,and the results show that the ROS burst,callose deposition accumulate with 167A treatment.Moreover,167A also influence the degree of stomal closed.RNA-seq assays show that the 167A down-regulated the ABA associated genes and upregulated the JA/SA/ET associated genes.Through genetic analysis,the 167A modulating the plant resistance through the PYR/PYL Receptors.Together,these results demonstrate that a novel ABA analogue 167A positive regulated plant immunity and has great potential for agricultural applications.

TIR domain protein-mediated phase separation activates plant immunity

We spotlight recent findings from a Nature paper unveiling captivating insights into how substrates such as NADþand ATP stimulate the condensation of TIR domain proteins.This process culminates in the formation of a quaternary structural pattern akin to the catalytic arrangement observed in conventional TNL proteins.Consequently,this mechanism enables the production of pivotal signaling molecules crucial for fortifying plant immunity.Expanding on these revelations,we propose the prospect of creating modulatory compounds capable of initiating the phase separation of TIR domain proteins as an innovative approach to enhance plant immunity against pathogenic challenges.

The RECEPTOR-LIKE PROTEIN53 immune complex associates with LLG1 to positively regulate plant immunity

Pattern recognition receptors(PRRs)sense ligands in pattern-triggered immunity(PTI).Plant PRRs include numerous receptor-like proteins(RLPs),but many RLPs remain functionally uncharacterized.Here,we examine an Arabidopsis thaliana RLP,RLP53,which positively regulates immune signaling.Our forward genetic screen for suppressors of enhanced disease resistance1(edr1)identified a point mutation in RLP53 that fully suppresses disease resistance and mildewinduced cell death in edr1 mutants.The rlp53 mutants showed enhanced susceptibility to virulent pathogens,including fungi,oomycetes,and bacteria,indicating that RLP53 is important for plant immunity.The ectodomain of RLP53 contains leucine-rich repeat(LRR)motifs.RLP53 constitutively associates with the LRR receptorlike kinase SUPPRESSOR OF BRASSINOSTEROIDINSENSITIVE1-ASSOCIATEDKINASE(BAK1)-INTERACTINGRECEPTORKINASE1(SOBIR1)and interacts with the co-receptor BAK1 in a pathogen-induced manner.The double mutation sobir1-12 bak1-5 suppresses edr1-mediated disease resistance,suggesting that EDR1 negatively regulates PTI modulated by the RLP53–SOBIR1–BAK1 complex.Moreover,the glycosylphosphatidylinositol(GPI)-anchored protein LORELEI-LIKE GPI-ANCHORED PROTEIN1(LLG1)interacts with RLP53 and mediates RLP53 accumulation in the plasma membrane.We thus uncovered the role of a novel RLP and its associated immune complex in plant defense responses and revealed a potential new mechanism underlying regulation of RLP immune function by a GPI-anchored protein.

RALF22 promotes plant immunity and amplifies the Pep3 immune signal

Rapid alkalinization factors(RALFs)in plants have been reported to dampen pathogenassociated molecular pattern(PAMP)-triggered immunity via suppressing PAMP-induced complex formation between the pattern recognition receptor(PRR)and its co-receptor BAK1.However,the direct and positive role of RALFs in plant immunity remains largely unknown.Herein,we report the direct and positive roles of a typical RALF,RALF22,in plant immunity.RALF22alone directly elicited a variety of typical immune responses and triggered resistance against the devastating necrotrophic fungal pathogen Sclerotinia sclerotiorum in a FERONIA(FER)-dependent manner.LORELEI(LRE)-like glycosylphosphatidylinositol(GPI)-anchored protein 1(LLG1)and NADPH oxidase RBOHD were required for RALF22-elicited reactive oxygen species(ROS)generation.The mutation of cysteines conserved in the C terminus of RALFs abolished,while the constitutive formation of two disulfide bridges between these cysteines promoted the RALF22-elicited ROS production and resistance against S.sclerotiorum,demonstrating the requirement of these cysteines in the functions of RALF22 in plant immunity.Furthermore,RALF22 amplified the Pep3-induced immune signal by dramatically increasing the abundance of PROPEP3 transcript and protein.Supply with RALF22 induced resistance against S.sclerotiorum in Brassica crop plants.Collectively,our results reveal that RALF22 triggers immune responses and augments the Pep3-induced immune signal in a FER-dependent manner,and exhibits the potential to be exploited as an immune elicitor in crop protection.

Systems understanding of plant-pathogen interactions through genome-wide protein-protein interaction networks

Plants are frequently affected by pathogen infections.To effectively defend against such infections,two major modes of innate immunity have evolved in plants;pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity.Although the molecular components as well as the corresponding pathways involved in these two processes have been identified,many aspects of the molecular mechanisms of the plant immune system remain elusive.Recently,the rapid development of omics techniques(e.g.,genomics,proteomics and transcriptomics) has provided a great opportunity to explore plant–pathogen interactions from a systems perspective and studies on protein–protein interactions(PPIs) between plants and pathogens have been carried out and characterized at the network level.In this review,we introduce experimental and computational identification methods of PPIs,popular PPI network analysis approaches,and existing bioinformatics resources/tools related to PPIs.Then,we focus on reviewing the progress in genome-wide PPI networks related to plant–pathogen interactions,including pathogen-centric PPI networks,plant-centric PPI networks and interspecies PPI networks between plants and pathogens.We anticipate genome-wide PPI network analysis will provide a clearer understanding of plant–pathogen interactions and will offer some new opportunities for crop protection and improvement.

From molecule to cell:the expanding frontiers of plant immunity

In recent years,the field of plant immunity has witnessed remarkable breakthroughs.During the co-evolution between plants and pathogens,plants have developed a wealth of intricate defense mechanisms to safeguard their survival.Newly identified immune receptors have added unexpected complexity to the surface and intracellular sensor networks,enriching our understanding of the ongoing plant–pathogen interplay.Deciphering the molecular mechanisms of resistosome shapes our understanding of these mysterious molecules in plant immunity.Moreover,technological innovations are expanding the horizon of the plant–pathogen battlefield into spatial and temporal scales.While the development provides new opportunities for untangling the complex realm of plant immunity,challenges remain in uncovering plant immunity across spatiotemporal dimensions from both molecular and cellular levels.

Near-infrared light and PIF4 promote plant antiviral defense by enhancing RNA interference

The molecular mechanism underlying phototherapy and light treatment,which utilize various wavelength spectra of light,including near-infrared(NIR),to cure human and plant diseases,is obscure.Here we re-vealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4(PIF4)-activated RNA interference(RNAi)in plants.PIF4,a central transcription factor involved in light signaling,accumulates to high levels under NIR light in plants.PIF4 directly induces the transcription of two essential components of RNAi,RNA-DEPENDENT RNA POLYMERASE 6(RDR6)and ARGONAUTE 1(AGO1),which play important roles in resistance to both DNA and RNA viruses.Moreover,the pathogenic determinant bC1 protein,which is evolutionarily conserved and encoded by betasatellites,interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization.Thesefindings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the explo-ration of NIR antiviral treatment.

CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

Rice genes OsDjA2 and OsERF104,encoding a chaperone protein and an APETELA2/ethylene-responsive factor,respectively,are strongly induced in a compatible interaction with blast fungus,and also have function in plant susceptibility validated through gene silencing.Here,we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance.A total of 15 OsDjA2(62.5%)and 17 OsERF104(70.8%)T_(0)transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments.Phenotyping of homozygous T1 mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area,compared with the infected control plants.Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease.

Twisting mode of supercoil leucine-rich domain mediates peptide sensing in FLS2–flg22–BAK1 complex

Plants and animals recognize microbial invaders by detecting pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs). This recognition plays a crucial role in plant immunity. The newly discovered protein in plants that responds to bacterial flagellin, i.e., flagellin-sensitive 2 (FLS2), is ubiquitously expressed and present in many plants. The association of FLS2 and BAK1, facilitated by a highly conserved epitope flg22 of flagellin, triggers such downstream immune responses as activated MAPK pathway and elevated reactive oxygen species (ROS) for bacterial defense and plant immunity. Here we study the intrinsic dynamics and conformational change of FLS2 upon the formation of the FLS2–flg22–BAK1 complex. The top intrinsic normal modes and principal structural fluctuation components are very similar, showing two bending modes and one twisting mode. The twisting mode alone, however, accounts for most of the conformational change of FLS2 induced by binding with flg22 and BAK1. This study indicates that flg22 binding suppresses FLS2 conformational fluctuation, especially on the twisting motion, thus facilitating FLS2–BAK1 interaction. A detailed analysis of this sensing mechanism may aid better design on both PRR and peptide mimetics for plant immunity.