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.

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.

New insight into Ca^(2+)-permeable channel in plant immunity

Calcium ions(Ca^(2+)) are crucial intracellular second messengers in eukaryotic cells. Upon pathogen perception, plants generate a transient and rapid increase in cytoplasmic Ca^(2+)levels, which is subsequently decoded by Ca^(2+)sensors and effectors to activate downstream immune responses. The elevation of cytosolic Ca^(2+)is commonly attributed to Ca^(2+)influx mediated by plasma membranelocalized Ca^(2+)–permeable channels. However, the contribution of Ca^(2+)release triggered by intracellular Ca^(2+)-permeable channels in shaping Ca^(2+)signaling associated with plant immunity remains poorly understood. This review discusses recent advances in understanding the mechanism underlying the shaping of Ca^(2+)signatures upon the activation of immune receptors, with particular emphasis on the identification of intracellular immune receptors as non-canonical Ca^(2+)-permeable channels. We also discuss the involvement of Ca^(2+)release from the endoplasmic reticulum in generating Ca^(2+)signaling during plant immunity.

Epigenetic regulation of plant immunity:from chromatin codes to plant disease resistance

Facing a deteriorating natural environment and an increasing serious food crisis,bioengineering-based breeding is increasing in importance.To defend against pathogen infection,plants have evolved multiple defense mechanisms,including pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)and effector-triggered immunity(ETI).A complex regulatory network acts downstream of these PTI and ETI pathways,including hormone signal transduction and transcriptional reprogramming.In recent years,increasing lines of evidence show that epigenetic factors act,as key regulators involved in the transcriptional reprogramming,to modulate plant immune responses.Here,we summarize current progress on the regulatory mechanism of DNA methylation and histone modifications in plant defense responses.In addition,we also discuss the application of epigenetic mechanism-based resistance strategies in plant disease breeding.

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.

UBP12/UBP13-mediated deubiquitination of salicylic acid receptor NPR3 suppresses plant immunity

Salicylic acid(SA),a defense hormone produced after pathogen challenge,is critical for plant immunity.Arabidopsis NONEXPRESSER OF PR GENES 1(NPR1)and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transduction.NPR1 functions as a transcriptional co-activator to promote defense gene expression,whereas NPR3 and NPR4 have been shown to function as negative regulators in the SA signaling pathway.Although the mechanism about NPR1 regulation has been well studied,how NPR3/NPR4 proteins are regulated in immune responses remains largely unknown.Here,we show that the stability of NPR3/NPR4 is enhanced by SA.In the absence of pathogen challenge,NPR3/NPR4 are unstable and degraded by the 26S proteasome,whereas the increase in cellular SA levels upon pathogen infection suppresses NPR3/NPR4 degradation.We found that UBP12 and UBP13,two homologous deubiquitinases from a ubiquitin-specific protease subfamily,negatively regulate plant immunity by promoting NPR3/NPR4 stability.Our genetic results further showed that UBP12/UBP13-mediated immunity suppression is partially dependent on NPR3/NPR4 functions.By interacting with NPR3 in the nucleus in an SA-dependent manner,UBP12 and UBP13 remove ubiquitin from polyubiquitinated NPR3 to protect it from being degraded.The stabilization of NPR3/NPR4 promoted by UBP12/UBP13 is essential for negative regulation of basal and SA-induced immunity.

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.

Plant immunity inducers:from discovery to agricultural application

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

Arabidopsis E3 ligase KEG associates with and ubiquitinates MKK4 and MKK5 to regulate plant immunity

Mitogen-activated protein kinase(MAPK) cascades are highly conserved signaling modules that regulate plant immune responses. The Arabidopsis thaliana Raf-like MAPK kinase kinase ENHANCED DISEASE RESISTANCE1(EDR1) is a key negative regulator of plant immunity that affects the protein levels of MKK4 and MKK5, two important MAPK cascade members, but the underlying mechanism is poorly understood. Here, genome-wide phosphorylation analysis demonstrated that the E3 ligase KEEP ON GOING(KEG) is phosphorylated in the edr1 mutant but not the wild type, suggesting that EDR1 negatively affects KEG phosphorylation. The identified phosphorylation sites in KEG appear to be important for its accumulation. The keg-4 mutant, a previously identified edr1 suppressor, enhances susceptibility to the powdery mildew pathogen Golovinomyces cichoracearum. In addition, MKK4 and MKK5 protein levels are reduced in the keg-4 mutant. Furthermore,we demonstrate that MKK4 and MKK5 associate with full-length KEG, but not with truncated KEG-RK or KEG-RKA, and that KEG ubiquitinates and mediates the degradation of MKK4 and MKK5. Taken together, these results indicate that MKK4 and MKK5 protein levels are regulated by KEG via ubiquitination, uncovering a mechanism by which plants finetune immune responses by regulating the homeostasis of key MAPK cascade members via ubiquitination and degradation.

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.

Crosstalk between Ubiquitination and Other Posttranslational Protein Modifications in Plant Immunity

Post-translational modifications(PTMs)are central to the modulation of protein activity,stability,subcellular localization,and interaction with partners.They greatly expand the diversity and functionality of the proteome and have taken the center stage as key players in regulating numerous cellular and physiological processes.Increasing evidence indicates that in addition to a single regulatory PTM,many proteins are modified by multiple different types of PTMs in an orchestrated manner to collectively modulate the biological outcome.Such PTM crosstalk creates a combinatorial explosion in the number of proteoforms in a cell and greatly improves the ability of plants to rapidly mount and fine-tune responses to different external and internal cues.While PTM crosstalk has been investigated in depth in humans,animals,and yeast,the study of interplay between different PTMs in plants is still at its infant stage.In the past decade,investigations showed that PTMs are widely involved and play critical roles in the regulation of interactions between plants and pathogens.In particular,ubiquitination has emerged as a key regulator of plant immunity.This review discusses recent studies of the crosstalk between ubiquitination and six other PTMs,i.e.,phosphorylation,SUMOylation,poly(ADP-ribosyl)ation,acetylation,redox modification,and glycosylation,in the regulation of plant immunity.The two basic ways by which PTMs communicate as well as the underlying mechanisms and diverse outcomes of the PTM crosstalk in plant immunity are highlighted.

From plant immunity to crop disease resistance

Plant diseases caused by diverse pathogens lead to a serious reduction in crop yield and threaten food security worldwide.Genetic improvement of plant immunity is considered as the most effective and sustainable approach to control crop diseases.In the last decade,our understanding of plant immunity at both molecular and genomic levels has improved greatly.Combined with advances in biotechnologies,particularly clustered regularly interspaced short palindromic repeat(CRISPR)/Cas9-based genome editing,we can now rapidly identify new resistance genes and engineer disease-resistance crop plants like never before.In this review,we summarize the current knowledge of plant immunity and outline existing and new strategies for disease resistance improvement in crop plants.We also discuss existing challenges in this field and suggest directions for future studies.

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3(MAPK3 or MPK3)and MPK6 play important signaling roles in plant immunity and growth/development.MAPK KINASE4(MKK4)and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes.YODA(YDA),also known as MAPK KINASE KINASE4(MAPKKK4),is upstream of MKK4/MKK5 and forms a complete MAPK cascade(YDA–MKK4/MKK5–MPK3/MPK6)in regulating plant growth and development.In plant immunity,MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5–MPK3/MPK6 module.However,the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern(PAMP)treatment suggests the presence of additional MAPKKK(s)in this MAPK cascade in signaling plant immunity.To investigate whether YDA is also involved in plant immunity,we attempted to generate mapkkk3 mapkkk5 yda triple mutants.However,it was not possible to recover one of the double mutant combinations(mapkkk5 yda)or the triple mutant(mapkkk3 mapkkk5 yda)due to a failure of embryogenesis.Using the clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPRassociated protein 9(Cas9)approach,we generated weak,N-terminal deletion alleles of YDA,yda-del,in a mapkkk3 mapkkk5 background.PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant,and the triple mutant was more susceptible to pathogen infection,suggesting YDA also plays an important role in plant immune signaling.In addition,MAPKKK5 and,to a lesser extent,MAPKKK3 were found to contribute to gamete function and embryogenesis,together with YDA.While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant,mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal,similar to the mpk3 mpk6 double mutants.These results demonstrate that YDA,MAPKKK3,and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5–MPK3/MPK6 module in both plant immunity and growth/development.

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization.Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection,the molecular basis of Avh241 virulence remains poorly understood.Here we identified non-race specific disease resistance 1(NDR1)-like proteins,the critical components in plant effector-triggered immunity(ETI)responses,as host targets of Avh241.Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses.Silencing of GmNDR1s increases the susceptibility of soybean to P.sojae infection,and overexpression of GmNDR1s reduces infection,which supports its positive role in plant immunity against P.sojae.Furthermore,we demonstrate that GmNDR1 interacts with itself,and Avh241 probably disrupts the self-association of GmNDR1.These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses,likely by disturbing the function of NDR1 during infection.

Calcium channels at the center of nucleotide-binding leucine-rich repeat receptor-mediated plant immunity

Higher plants utilize a variety of immune receptors to recognize pathogens and trigger defense responses.Intracellular nucleotidebinding leucine-rich repeat receptors(NLRs)are widely used for detecting pathogen effectors(Jones et al.,2016;Zhou and Zhang,2020).NLRs are also present in animals,including mammals。

Phytocytokines function as immunological modulators of plant immunity

Plant plasma membrane-resident immune receptors regulate plant immunity by recognizing microbe-associated molecular patterns(MAMPs),damage-associated molecular patterns(DAMPs),and phytocytokines.Phytocytokines are plant endogenous peptides,which are usually produced in the cytosol and released into the apoplast when plant encounters pathogen infections.Phytocytokines regulate plant immunity through activating an overlapping signaling pathway with MAMPs/DAMPs with some unique features.Here,we highlight the current understanding of phytocytokine production,perception and functions in plant immunity,and discuss how plants and pathogens manipulate phytocytokine signaling for their own benefits during the plant-pathogen warfare.

Plant NLRs: The Whistleblowers of Plant Immunity

The study of plant diseases is almost as old as agriculture itself. Advancements in molecular biology havegiven us much more insight into the plant immune system and how it detects the many pathogens plantsmay encounter. Members of the primary family of plant resistance (R) proteins, NLRs, contain three distinctdomains, and appear to use several different mechanisms to recognize pathogen effectors and trigger immunity. Understanding the molecular process of NLR recognition and activation has been greatly aided byadvancements in structural studies, with ZAR1 recently becoming the first full-length NLR to be visualized.Genetic and biochemical analysis identified many critical components for NLR activation and homeostasiscontrol. The increased study of helper NLRs has also provided insights into the downstream signaling pathways of NLRs. This review summarizes the progress in the last decades on plant NLR research, focusing onthe mechanistic understanding that has been achieved.

Arabidopsis CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant immunity

Arabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1(SARD1)and CALMODULIN-BINDING PROTEIN 60g(CBP60g)are two master transcription factors that regulate many defense-related genes in plant immunity.They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1,CONSTITUTIVE 2(SNC2).Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected in either sard1 or cbp60g single mutants but completely suppressed in the sard1 cbp60g double mutant.Here we report that CBP60b,another member of the CBP60 family,also functions as a positive regulator of SNC2-mediated immunity.Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D,whereas overexpression of CBP60b leads to elevated SARD1 expression and constitutive defense responses.In addition,transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene.Chromatin immunoprecipitation assays further showed that CBP60b is recruited to the promoter region of SARD1,suggesting that it directly regulates SARD1 expression.Interestingly,knocking out CBP60b in the wild-type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1(EDS1)-dependent autoimmunity,suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator of immunity mediated by receptors carrying an N-terminal Toll-interleukin-1 receptor-like domain.

EDS1 modules as two‑tiered receptor complexes for TIR‑catalyzed signaling molecules to activate plant immunity

Plant intracellular nucleotide-binding leucine-rich repeat(NLR)receptors with an N-terminal Toll/Interleukin-1 recep-tor(TIR)domain detect pathogen effectors to produce TIR-catalyzed signaling molecules for activation of plant immunity.Plant immune signaling by TIR-containing NLR(TNL)proteins converges on Enhanced Disease Suscepti-bility 1(EDS1)and its direct partners Phytoalexin Deficient 4(PAD4)or Senescence-Associated Gene 101(SAG101).TNL signaling also require helper NLRs N requirement gene 1(NRG1)and activated disease resistance 1(ADR1).In two recent remarkable papers published in Science,the authors show that the TIR-containing proteins catalyze and produce two types of signaling molecules,ADPr-ATP/diADPR and pRib-AMP/ADP.Importantly,they demonstrate that EDS1-SAG101 and EDS1-PAD4 modules are the receptor complexes for ADPr-ATP/diADPRp and Rib-AMP/ADP,respec-tively,which allosterically promote EDS1-SAG101 interaction with NRG1 and EDS1-PAD4 interaction with ADR1.Thus,two different small molecules catalyzed by TIR-containing proteins selectively activate the downstream two distinct branches of EDS1-mediated immune signalings.These breakthrough studies significantly advance our understanding of TNL downstream signaling pathway.