[目的]为明确几丁质和鞭毛蛋白衍生肽flg22诱导的辣椒幼苗先天免疫生理响应特征,探讨辣椒先天免疫生理响应与辣椒抗多种病害的关系.[方法]以5个四川本地辣椒品种幼苗为试材,鉴定它们对青枯病和疫病病情指数和抗性水平,采用水培法培育幼苗并进行外源几丁质和flg22处理,检测各品种不同诱导时间下幼苗根系生长、气孔孔径、胼胝质沉积、活性氧(ROS)积累和SOD、CAT活性,以及先天免疫相关基因表达量的变化,综合评价其生理响应及其与抗病性关系.[结果](1)各品种幼苗青枯病和疫病病情指数以'川腾10号'最低,抗病性最强,以'本地条椒'最高,抗病性最弱.(2)外源几丁质和flg22抑制了各品种幼苗根系生长速率,诱导离体叶片气孔闭合,促进叶片细胞壁胼胝质沉积加厚,ROS含量持续增加,SOD和CAT活性不断提高.各品种幼苗先天免疫生理响应指标的平均隶属函数值以'川腾10号'最高,'本地条椒'最低,并且平均隶属函数值与疫病、青枯病病情指数均具有显著负相关性.(3)外源flg22和几丁质诱导'川腾10号'幼苗先天免疫相关基因CaWRKY22、CaMAPK7和ChiIV3显著上调表达.[结论]外源flg22和几丁质可诱导辣椒幼苗先天免疫生理响应,且响应程度强弱在品种间存在差异,依据生理响应指标通过隶属函数可综合评价辣椒品种的抗病水平;'川腾10号'的平均隶属函数值最高,多抗性水平最好,这与其先天免疫相关基因CaWRKY22、CaMAPK7和ChiIV3的显著上调表达有关.

Pattern-triggered immunity(PTI)and effector-triggered immunity(ETI)are required for host defense against pathogens.Although PTI and ETI are intimately connected,the underlying molecular mechanisms remain elusive.In this study,we demonstrate that flg22 priming attenuates Pseudomonas syringae pv.to-mato DC3000(Pst)AvrRpt2-induced hypersensitive cell death,resistance,and biomass reduction in Arabi-dopsis.Mitogen-activated protein kinases(MAPKs)are key signaling regulators of PTI and ETI.The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression(PES).We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18,which regulates the expression of AP2C1 and PP2C5,two genes encoding protein phosphatases.Furthermore,we observed that the PTI-suppressed ETI-triggered cell death,MAPK activation,and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants.Taken together,our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

Mitogen-activated protein kinase(MAPK)cas-cades play pivotal roles in plant defense against phytopathogens downstream of im-mune receptor complexes.The amplitude and duration of MAPK activation must be strictly controlled,but the underlying mechanism re-mains unclear.Here,we identified Arabidopsis CPL1(C-terminal domain phosphatase-like 1)as a negative regulator of microbe-associated molecular pattern(MAMP)-triggered immunity via a forward-genetic screen.Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bac-terial peptide flg22.Furthermore,flg22-induced MPK3/MPK4/MPK6 phosphorylation was dra-matically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines,sug-gesting that CPL1 might interfere with flg22-induced MAPK activation.Indeed,CPL1 di-rectly interacted with MPK3 and MPK6,as well as the upstream MKK4 and MKK5.A firefly luciferase-based complementation assay in-dicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly re-duced in the presence of CPL1.These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

Cell-surface-localized leucine-rich-repeat receptor-like kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana ben-thamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly com-promised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phy-tophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROID-SIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced im-munity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results sug-gest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 asso-ciation is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes.Serine pal-mitoyltransferase(SPT)is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway.The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1(LCB1)and LCB2 subunits.SPT activity is in-hibited by orosomucoid proteins and stimulated by small subunits of SPT(ssSPTs).However,whether LCB1 is modified and how such mod-ification might regulate SPT activity have to date been unclear.Here,we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3(MPK3)and MPK6 by upstream MKK9 and treat-ment with Flg22(a pathogen-associated molec-ular pattern)increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana,with activated MPK3 and MPK6 phosphorylating AtLCB1.Phosphor-ylation of AtLCB1 strengthened its binding with AtLCB2b,promoted its binding with ssSPTs,and stimulated the formation of higher order oligo-meric and active SPT complexes.Our findings therefore suggest a novel regulatory mechanism for SPT activity.

Arabidopsis heterotrimeric G proteins regulate diverse processes by coupling to single-transmembrane receptors. One such receptor is the FLS2 receptor kinase, which perceives bacterial flagellin epitope flg22 to activate immunity through a class of cytoplasmic kinases called BIK1/PBLs. Unlike animal and fungal heterotrimeric G proteins that are activated by a ligand-induced guanine nucleotide exchange activity of seven-transmembrane G protein-coupled receptors (GPCRs), plant heterotrimeric G proteins are self-activating. How plant receptors regulate heterotrimeric G proteins in response to external ligands remains unknown. Here we show that RGS1, a GTPase accelerating protein, maintains Arabidopsis G proteins in an inactive state in complex with FLS2. Activation of FLS2 by flg22 induces a BIK1/PBL-mediated phosphorylation of RGS1 at Ser428 and Ser431 and that promotes RGS1 dissociation from the FLS2-G protein complex. This relieves G proteins from the RGS1-mediated repression and enables positive regulation of immune signaling. We additionally show that RGS1 is similarly regulated by multiple immune receptors. Our results uncover ligand-induced de-repression as a mechanism for G protein signaling in plants that is distinct from previously reported mechanism underlying the activation of heterotrimeric G proteins in other systems.

绿色植物中的快速碱化因子(Rapid alkalinization factor,RALF)为一类进化保守的多肽信号分子,以基因家族形式存在.模式植物拟南芥中至少存在35个RALF基因成员,前期研究显示拟南芥RALF家族的部分成员,比如RALF1/23,RALF4/19可分别作为CrRLK1L类蛋白受体激酶家族成员FERONIA及BUPS1/2的配体,调控细胞伸长、植物免疫应答及双受精等过程,但是RALF家族其他成员是否具有生物学活性,以及不同成员之间是否具有功能性差异均尚不清楚.因此,本研究异源表达了19个代表性的RALF,并对其生物学活性和功能性差异进行了分析.实验结果表明,19个RALF均对根的生长起到不同程度的抑制作用,进一步挑选了部分代表性RALF成员进行了活性氧(Reactive oxygen species,ROS)迸发及丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)磷酸化实验分析,实验结果表明,我们确证了11个RALF蛋白参与了MAPK信号的响应,同时,证实16个RALF蛋白抑制了由flg22引起的ROS的释放.此外,不同RALF成员在下胚轴细胞伸长上的作用也存在明显差异,比如RALF10促进下胚轴的伸长.以上研究结果表明不同RALF之间既存在功能冗余性,又存在功能性差异.本研究丰富了对RALF功能复杂性和多样性的认识.

Plants can produce animal cytokine-like immune peptides,among which plant elicitor peptides(Peps)derive from the C termini of their precursors(PROPEPs).Recently,the functions of Peps have been expanded beyond plant immunity.However,a long-standing enigma is how PROPEPs are processed into Peps.Here,we report that the Ca2+-dependent type-Ⅱ metacaspases(MCs)constitute the proteolytic enzymes to mediate PROPEP processing in Arabidopsis.In protoplasts,co-expression of PROPEP1 with type-Ⅱ MCs,including MC4 to MC9,can promote the generation of processed Pep1.Destruction of the cat-alytic cysteine residue in MC4 or the conserved arginine residue preceding the Pep1 sequence blocks PRO-PEP1 cleavage,whereas the bacterial elicitor flg22 enhances the MC4-mediated PROPEP1 processing.MC4 cleaves PROPEP1 in vitro and also cleaves PROPEP2 to PROPEP8,but,surprisingly,not PROPEP6 in protoplasts.Domain swapping between PROPEP1 and PROPEP6 suggests a hidden role of the sequence context upstream of the Pep sequence for PROPEP processing.flg22-induced PROPEP1 processing and Botrytis cinerea resistance are severely impaired in the mc4/5/6/7 quadruple-mutant plants.Taken together,our study identifies the type-Ⅱ MCs as new players in Pep signaling,and lays the foundation for understanding the regulation of multifaceted functions of Peps in plant immunity and beyond.

Heterotrimeric G proteins consisting of Gα,Gβand Gγ are conserved signaling hubs in eukaryotes.Without analogs to canonical animal G protein-coupled receptors,plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα.Meanwhile,the exact role of canonical Gcα in plant innate immunity remains controversial.Here,we report multiple immune deficiencies in the null allele of Arabidopsis Gα(GPA1) in response to bacterial fig22 elicitor,clarifying a positive regulatory role of GPA1 in fig22 signaling.We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr19 upon flg22 elicitation.Interestingly,fig22 could not induce phosphorylation of GPA1T19A and GPA1T19D,suggesting that the dynamic Thr19 phosphorylation is required for GPA1 to respond to fig22.Moreover,flg22-induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo,and BAK1 could phosphorylate GPA1 but not GPA1T19A in vitro at the phosphorylation sites identified in vivo,suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to fig22.Furthermore,the T1gA mutation could promote flg22-induced association,rather than dissociation,between GPA1 and RGS1.Taken together,our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.

Plant cells mount plenty of pattern-recognition receptors (PRRs) to detect the microbe-associated molec-ular patterns (MAMPs) from potential microbial pathogens. MAMPs are overrepresented by proteinaneous patterns, such as the flg22 peptide from bacterial flagellin. Identi-fication of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming, and be confounded by functional redundancies. Here, we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged pro-teinaneous MAMPs to the apoplast. The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spec-trometry. As a proof of principle, we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiol@induction. Moreover, we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex, which were further validated by pro-tein–protein interaction assays and the reverse genetics approach. Our study showcases a simple way to bio-chemically identify endogenous PRR complex components without overexpressing the PRR or using chemical cross-linkers, and suggests a possible crosstalk between different immune receptors in plants. A modest dose of estradiol can also be applied to inducing enhanced immunity in en-gineered plants to both bacterial and fungal pathogens.