Identif ication of a hypersensitive response core peptide of HrpZ and its role in increasing grape downy mildew resistance

Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.

Alpha-picolinic acid,a fungal toxin and mammal apoptosis-inducing agent,elicits hypersensitive-like response and enhances disease resistance in rice

Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here,we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS),rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride,indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence,it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice,which also provides a simple eliciting tool for studying apoptosis in the plant cell.

Disruption of non-classically secreted protein(MoMtp)compromised conidiation,stress homeostasis,and pathogenesis of Magnaporthe oryzae

Blast disease,caused by the hemibiotrophic ascomycete fungus,Magnaporthe oryzae,is a significant threat to sustainable rice production worldwide.Studies have shown that the blast fungus secretes vast arrays of functionally diverse proteins into the host cell for a successful disease progression.However,the final destinations of these effector proteins inside the host cell and their role in advancing fungal pathogenesis remain a mystery.Here,we reported that a putative mitochondrial targeting non-classically secreted protein(MoMtp)positively regulates conidiogenesis and appressorium maturation in M.oryzae.Moreover,MoM TP gene deletion mutant strains triggered a hypersensitive response when inoculated on rice leaves displaying that MoMtp is essential for the virulence of M.oryzae.In addition,cell wall and oxidative stress results indicated that MoMtp is likely involved in the maintenance of the structural integrity of the fungus cell.Our study also demonstrates an upregulation in the expression pattern of the MoMTP gene at all stages of infection,indicating its possible regulatory role in host invasion and the infectious development of M.oryzae.Furthermore,Agrobacterium infiltration and sheath inoculation confirmed that MoMtpGFP protein is predominantly localized in the host mitochondria of tobacco leaf and rice cells.Taken together,we conclude that MoMtp protein likely promotes the normal conidiation and pathogenesis of M.oryzae and might have a role in disturbing the proper functioning of the host mitochondria during pathogen invasion.

OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice

The basic region/leucine zipper(bZIP)transcription factors play important roles in plant development and responses to abiotic and biotic stresses.OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants.To further investigate the biological functions of OsbZIP53,we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants.Comprehensive analysis of phenotypical,physiological,and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants,but also regulated the immune response through the salicylic acid pathway.Specifically,disrupting OsbZIP53 increased H2O2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs(Osrboh genes)and weakened H2O2 degradation by directly targeting OsMYBS1.In addition,the growth of osbzip53 mutants was seriously impaired,while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type,suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

Structural and Functional Insights into an Arabidopsis NBS-LRR Receptor in Nicotiana benthamiana

Nucleotide-binding site leucine-rich repeat receptors (NBS-LRR/NLRs) are crucial intracellular immune proteins in plants. Previous article reported a novel NLR protein SUT1 (SUPPRESSORS OF TOPP4-1, 1), which is involved in autoimmunity initiated by type one protein phosphatase 4 mutation (topp4-1) in Arabidopsis, however, its role in planta is still unclear. This study employed Nicotiana benthamiana, a model platform, to conduct an overall structural and functional analysis of SUT1 protein. The transient expression results revealed that SUT1 is a typical CNL (CC-NBS-LRR) receptor, both fluorescence data and biochemical results showed the protein is mainly anchored on the plasma membrane due to its N-terminal acylation site. Further truncation experiments announced that its CC (coiled-coil) domain possessed cell-death-inducing activity. The outcomes of point mutations analysis revealed that not only the CC domain, but also the full-length SUT1 protein, whose function and subcellular localization are influenced by highly conserved hydrophobic residues. These research outcomes provided favorable clues for elucidating the activation mechanism of SUT1.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

Induction of Hypersensitive Response and Nonhost Resistance by a Cladosporium fulvum Elicitor CfHNNI1 is Dose-Dependent and Negatively Regulated by Salicylic Acid

Nonhost resistance is a phenomenon that enables plants to protect themselves against the majority of potential pathogens, and thus has a great potential for application in plant protection. We recently found that CfHNNI1 （for Cladosporium fulvum host and nonhost plant necrosis inducer 1） is an inducer of plant hypersensitive response （HR） and nonhost resistance. In this study, its functional mechanism was analyzed. CfHNN11 was a single copy gene in C. fulvum genome. The functional ORF of the CfHNN11 cDNA was ATG3-TAG780, which showed homology with genes encoding bZIP transcription factors. The functional ORF included in frame an inner one ATG273-TAG780, which was sufficient to induce HR in plants. CfIINN11 induced plant HR in a dose-dependent manner. CfHNNIl-induced necrosis in NahG transgenic tomato plants was significantly stronger than that in their wild type controls. However, the necrosis in Nr and defl tomato mutants was similar to that in their corresponding wild type plants. These data demonstrate that induction of HR and nonhost resistance by CfHNNI1 is negatively regulated by salicylic acid signalling pathway but independent of ethylene and jasmonic acid signalling pathways.

Physical Mapping of the Sequences Homologous to Disease Resistance Genes myb1 and NDR1 in Maize

Using fluorescence in situ hybridization, the authors investigated the homology between three plant species, maize (Zea mays L.) and tobacco (Nicotiana tabacum L.), maize and Arabidopsis thaliana (L.) Heynh. at cytogenetic level using two probes corresponding to functional disease resistance genes myb1 and NDR1 in Arabidopsis and tobacco respectively. The hybridization signals of the tested probes were detected in maize chromosomes 8 and 5 respectively, and the single location of each of the two probes showed only single copy of them in maize genome. The results provided a valuable insight into searching for genes associated with programmed cell death in plants using heterologous probe with comparative genetic approach. In addition, the improvements of FISH technique using heterologous probes were discussed.

Avr/Cf Interaction Induce the Expression of Ptis, Genes Encoding Pto-interactors

Products of plant resistance ( R ) genes Pto and Cf contain distinct domains, and have different cellular localization. It is intriguing to compare the development mechanisms of resistance conferred by the two R genes. In the present report, two hypersensitive response (HR) initiation systems were employed to study the time_course expression induced by Avr / Cf interaction of the genes encoding Pti4, Pti5 and Pti6 which interact directly with Pto: (1) Seeds of tomato (Lycopersicon esculentum Mill.) containing complementary gene pairs Avr 4/ Cf _4 and Avr 9/ Cf _9 were obtained through crossing. Their seedlings developed HR under room temperature. (2) Avr / Cf seedlings grew normally at 33 ℃. When the temperature was shifted down to 25 ℃, HR occurred within hours in the seedlings. Results of both experiments showed that expression of Pti4, Pti5 and Pti6 was induced upon development of hypersensitive necrosis in Avr / Cf seedlings. However, the expression levels and patterns of these Pti s differed. This finding indicated that these Pti s function complementarily, and might be involved in regulation of both Pto and Cf _conferred resistance.

Xoryp_08180 of Xanthomonas oryzae pv.oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.

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.

Using a Suppression Subtractive Library-Based Approach to Identify Non-Heading Chinese Cabbage Genes Up-Regulated in Early Response to Elicitor PB90

Monitoring expression at the transcriptional level is the first essential step for the functional analysis of plant genes. Genes-encoding proteins directly involved in early response to elicitor constitute only a small fraction of all the genes affected by elicitor. Transcriptional responses to various elicitors have been extensively studied in different plants including Nicotiana and Arabidopsis thaliana; however, corresponding data aren＇t available for non-heading Chinese cabbage. To address this problem, we describe a suppression subtractive library-based approach to isolate the plant＇s ESTs up-regulated in the early induction/execution of the HR induced by elicitor PB90 from Phytophthora boehmeriae. According to their putative identification in BLAST searches against the three genome databases, 70 up-regulated genes were classified into 9 parts： some aspect of primary ＇metabolism＇ or ＇energy＇ production; ＇protein synthesis＇ or ＇protein fate＇; cellular communication/signal transduction mechanism; cell fates including Beclin, SPT1, and SPT2; HLA-B and AGO1 which participate in transcription; cellular transport and hypothetical proteins or proteins for which a function has yet to be determined. Seven selected genes such as Beclin, thioredoxin, HLA-B, MAP3K, SPT1, SPT2, and AGO1 were up-regulated induced by PB90, suggesting that the genes may play an important role in PB90-triggered HR.

Hypersensitivity of vascular alpha-adrenoceptor responsiveness: a possible inducer of pain in neuropathic states

Dear editor,Dr. Peter Drummond＇s article noted that peripheral nerve and tissue injury in neuropathic pain syndromes releases cytokines which in turn lead to an increase in alphal-adrenoceptor upreg- ulation, resulting in a heightened sensitivity to noradrenaline. In these circumstances, noradrenaline acting on upregulated alpha1 a-adrenoceptors increases the release of cytokine interleukin-6. Hence, nociceptive afferent neurons exposed to injury induced cytokines become more hypersensitive to noradrenaline, which in turn promotes the release of more inflammatory cytokines. Dr. Drummond noted that this mechanism may contribute to the pain of post-herpetic neuralgia or complex regional pain syn- drome （Drummond, 2014）.

Erythema Nodosum Manifestation Post COVID-19 Vaccine: A Case Report

Erythema nodosum (EN) is a delayed hypersensitivity response that may be triggered by a range of conditions, including infections and vaccines. Rare cases of EN caused by COVID-19 were recently reported but none due to COVID-19 vaccines were documented. We report here a case of EN occurring after COVID-19 vaccination. Patient presented with painful nodular lesions of all 4 limbs, evolving for one month. These lesions appeared 48 h after the second dose of COVID-19 vaccination. The patient reported no recent infectious episodes. The physical examination found numerous, erythematous dermohypodermatitis knots with no palpable adenopathy. Some were regressive according to biligenesis shades. Biology and radiology findings eliminated other common causes of this dermatosis. The skin biopsy was done and suggested EN. The final diagnosis was post COVID-19 vaccine EN. Patient received a symptomatic treatment and had a slight improvement of the lesions 10 days after diagnosis. Physicians should be aware of the side effects of the vaccine including skin manifestations, especially since more people are bound to be vaccinated.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice

A key event that follows pathogen recognition by a resistance(R)protein containing an NB-ARC(nucleotide-binding adaptor shared by Apaf-1,R proteins,and Ced-4)domain is hypersensitive response(HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide.However,the integral mechanisms that underlie this process remain relatively opaque.Here,we show that a gain-offunction mutation in the NB-ARC protein RLS1(Rapid Leaf Senescence 1)triggers high-light-dependent HR-like cell death in rice.The RLS1-mediated defense response is largely independent of salicylic acid accumulation,NPR1(Nonexpressor of Pathogenesis-Related Gene 1)activity,and RAR1(Required for Mla12 Resistance 1)function.A screen for suppressors of RLS1 activation identified RMC(Root Meander Curling)as essential for the RLS1-activated defense response.RMC encodes a cysteine-rich receptor-like secreted protein(CRRSP)and functions as an RLS1-binding partner.Intriguingly,their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1.Collectively,our findings reveal an NBARC-CRRSP signaling module that modulates oxidative state,the cell death process,and associated immunity responses in rice.

H_2O_2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species

In plants, the chloroplast is the main reactive oxygen species （ROS） producing site under high light stress. Catalase （CAT）, which decomposes hydrogen peroxide （H2O2）, is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H2O2-induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 （noe1）, higher H2O2 levels induced the generation of nitric oxide （NO） and higher S-nitrosothiol （SNO） levels, suggesting that NO acts as an important endogenous mediator in H2O2-induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species （RNS）. Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H2O2-induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response （HR）, leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus(TYLCV)is known to encode 6 canonical viral proteins.Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions.The fifth ORF of TYLCV in the complementary sense,which we name C5,is evolutionarily conserved,but little is known about its expression and function during viral infection.Here,we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody.Ectopic expression of C5 using a potato virus X(PVX)vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species(ROS)in Nicotiana benthamiana plants.C5 was able to effectively suppress local and systemic post-transcriptional gene silencing(PTGS)induced by single-stranded GFP but not double-stranded GFP,and reversed the transcriptional gene silencing(TGS)of GFP.Furthermore,the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants.Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5.Collectively,this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor,hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

A Xanthomonas transcription activator-like effector is trapped in nonhost plants for immunity

Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial leaf blight in rice,delivers transcription activator-like effector(TALE)proteins into host cells to activate susceptibility or resistance(R)genes that promote disease or immunity,respectively.Nonhost plants serve as potential reservoirs of R genes;consequently,nonhost R genes may trap TALEs to trigger an immune response.In this study,we screened 17 Xoo TALEs for their ability to induce a hypersensitive response(HR)in the nonhost plant Nicotiana benthamiana(Nb);only AvrXa10 elicited an HR when transiently expressed in Nb.The HR generated by AvrXa10 required both the central repeat region and the activation domain,suggesting a specific interaction between AvrXa10 and a potential R-like gene in nonhost plants.Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death,and the transient expression of AvrXa10 in Nb induced immune responses.Genes targeted by AvrXa10 in the Nb genome were identified by transcriptome profiling and prediction of effector binding sites.Using several approaches(in vivo reporter assays,electrophoretic mobility-shift assays,targeted designer TALEs,and on-spot gene silencing),we confirmed that AvrXa10 targets NbZnFP1,a C2H2-type zinc finger protein that resides in the nucleus.Functional analysis indicated that overexpression of NbZnFP1 and its rice orthologs triggered cell death in rice protoplasts.An NbZnFP1 ortholog was also identified in tomato and was specifically activated by AvrXa10.These results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 to promote plant immunity in Nb.

ANNEXIN 8 negatively regulates RPW8.1-mediated cell death and disease resistance in Arabidopsis

Study on the regulation of broad-spectrum resistance is an active area in plant biology. RESISTANCE TO POWDERY MILDEW 8.1(RPW8.1)is one of a few broad-spectrum resistance genes triggering the hypersensitive response(HR) to restrict multiple pathogenic infections. To address the question how RPW8.1 signaling is regulated,we performed a genetic screen and tried to identify mutations enhancing RPW8.1-mediated HR.Here, we provided evidence to connect an annexin protein with RPW8.1-mediated resistance in Arabidopsis against powdery mildew. We isolated and characterized Arabidopsis b7-6 mutant.A point mutation in b7-6 at the At5 g12380 locus resulted in an amino acid substitution in ANNEXIN8(AtANN8). Loss-of-function or RNA-silencing of AtANN8 led to enhanced expression of RPW8.1,RPW8.1-dependent necrotic lesions in leaves, and defense against powdery mildew. Conversely,over-expression of AtANN8 compromised RPW8.1-mediated disease resistance and cell death. Interestingly, the mutation in AtANN8 enhanced RPW8.1-triggered H2O2. In addition, mutation in AtANN8 led to hypersensitivity to salt stress. Together, our data indicate that AtANN8 is involved in multiple stress signaling pathways and negatively regulates RPW8.1-mediated resistance against powdery mildew and cell death, thus linking ANNEXIN’s function with plant immunity.

Robust transcriptional indicators of immune cell death revealed by spatiotemporal transcriptome analyses

Recognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response(HR).This type of cell death occurs precisely at the site of pathogen recognition,and it is restricted to a few cells.Extensive research has shed light on how plant immune receptors are mechanistically activated.However,two central key questions remain largely unresolved:how does cell death zonation take place,and what are the mechanisms that underpin this phenomenon?Consequently,bona fide transcriptional indicators of HR are lacking,which prevents deeper insight into its mechanisms before cell death becomes macroscopic and precludes early or live observation.In this study,to identify the transcriptional indicators of HR we used the paradigmatic Arabidopsis thaliana–Pseudomonas syringae pathosystem and performed a spatiotemporally resolved gene expression analysis that compared infected cells that will undergo HR upon pathogen recognition with bystander cells that will stay alive and activate immunity.Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes,expression profiles,and biological processes derived from tissue undergoing HR and that of its surroundings.Furthermore,we generated a pipeline based on concatenated pairwise comparisons between time,zone,and treatment that enabled us to define 13 robust transcriptional HR markers.Among these genes,the promoter of an uncharacterized AAA-ATPase was used to obtain a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death.This valuable set of genes can be used to define cells that are destined to die upon infection with HR-triggering bacteria,opening new avenues for specific and/or high-throughput techniques to study HR processes at a single-cell level.