Hypersensitive Response Cell Death Research Articles

Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca2+ Influx and Ferroptotic Cell Death during Rice Immune Responses.

Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca2+ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca2+, ROS, and Fe2+ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, and H2O2 accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca2+ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca2+ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase.

Chemical-inducible gene expression systems are commonly used to regulate gene expression for functional genomics in various plant species. However, a convenient system that can tightly regulate transgene expression in Nicotiana benthamiana is still lacking. In this study, we developed a tightly regulated copper-inducible system that can control transgene expression and conduct cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system exhibited the least concerns regarding leakiness in N. benthamiana. Although the copper-inducible system can control the expression of some tested reporters, it is not sufficiently tight to regulate certain tested hypersensitive cell death responses. Using the MoClo-based synthetic biology approach, we incorporated the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested leucine-rich repeat-containing proteins and their matching avirulence factors, and it can be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings offer new approaches for both fundamental and translational studies in plant functional genomics.

Cytoplasmic Ca2+ influx mediates iron- and reactive oxygen species-dependent ferroptotic cell death in rice immunity.

Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca2+ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca2+ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). During Magnaporthe oryzae infection, 14 different Ca2+ influx regulators altered Ca2+, ROS and Fe2+ accumulation, glutathione reductase (GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca2+ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro. Ca2+ chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetra-acetic acid (EGTA) suppressed apoplastic Ca2+ influx in rice leaf sheaths during infection. Blocking apoplastic Ca2+ influx into the cytoplasm by Ca2+ chelation effectively suppressed Ca2+-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca2+ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.

Intracellular reactive oxygen species (intraROS)-aided localized cell death contributing to immune responses against wheat powdery mildew pathogen.

Reactive oxygen species (ROS) and hypersensitive response (HR) mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat pathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes (R genes), at a series of time points post-infection. The proportion of apoROS accumulation was 70-80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses were detected in 11-15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat (NLR) R genes (e.g. Pm3F, Pm41, TdPm60, MIIW72, Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed very less intraROS responses, while 11% of Pm24 line infected epidermis cells still showed HR cell death, suggesting that different resistance pathways are activated there. Here, we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat, although it induced the expression of pathogenesis-related (PR) genes. These results provide new insights on the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.

HexR Transcription Factor Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Coordinating Type Three Secretion System Genes.

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. We previously conducted a screening for reduced virulence using Tn5 transposon mutants and identified one of the transcriptional factors, HexR, as a potential Pcal virulence factor. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. Here, we demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We used RNA-seq analysis to characterize the genes regulated by HexR. We found that several type three secretion system (T3SS)-related genes had lower expression of the Pcal hexR mutant. Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es). We also confirmed that T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were also down-regulated in the Pcal hexR mutant both in culture and in vivo by using RT-qPCR. T3SS functions to suppress plant defense in host plants and induce hypersensitive response (HR) cell death in non-host plants. Therefore, we investigated the expression profiles of cabbage defense-related genes, including PR1 and PR5, and found that the expressions of these genes were greater in the Pcal hexR mutant. We also demonstrated that the hexR mutant did not induce HR cell death in non-host plants, indicating that HexR contributes in causing HR in nonhost plants. Together, these results indicate that the mutation in hexR leads to a reduction in the T3SS-related gene expression and thus an impairment in plant defense suppression, reducing Pcal virulence.

Two ways to die: Species dependent PCD modes in grapevine cells

Programmed cell death (PCD) is considered as a hallmark of strain-specific immunity. In contrast, generic basal immunity is thought to act without PCD. This classical bifurcation has been questioned during recent years. Likewise, the role of jasmonate signalling for these two modes of innate immunity has remained ambiguous. We have addressed both questions using two closely related grapevine cell lines (V. rupestris, V. vinifera cv. ‘Pinot Noir’) that contrast in their cell-death response to the bacterial elicitor harpin and the hormonal trigger methyl jasmonate (MeJA). We follow different cellular (loss of membrane integrity, mortality), molecular (induction of transcripts for phytoalexin synthesis and for metacaspases), as well as metabolic (sphingolipid profiles) responses to the two triggers in the two cell lines. The role of NADPH oxidases and induction of transcripts for the class-II metacaspases MC5 differ qualitatively between the two cell lines. We tested a possible role of sphingolipid metabolism but can rule this out. We propose a model, where V. rupestris, originating from co-evolution with several biotrophic pathogens, readily activates a hypersensitive cell death in response to harpin, while the context of MeJA-induced cell death in ‘Pinot Noir’ might not be related to immunity at all. We propose that the underlying signalling is modular, recruiting metacaspases differently depending on upstream signalling.

Candidatus Phytoplasma ziziphi encodes non-classically secreted proteins that suppress hypersensitive cell death response in Nicotiana benthamiana

Increasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.

Suppressed expression of ErbB3-binding protein 1 (EBP1) genes compromised the hypersensitive response cell death in Nicotiana benthamiana.

Target of rapamycin (TOR) regulates essential processes associated with plant growth, development, and cell death by modulating metabolic activities and translation in response to environmental signals. The ATP-competitive TOR inhibitor AZD8055 suppressed the hypersensitive response (HR) cell death in Nicotiana benthamiana infected with the incompatible Ralstonia solanacearum. The induced expression of the HR marker gene hin1 was also inhibited by the AZD8055 treatment. To further clarify the mechanisms underlying TOR-regulated HR cell death, we focused on TOR-related ErbB3-binding protein 1 (EBP1) in N. benthamiana (NbEBP1). We found four EBP1 orthologs in the N. benthamiana genome. The expression levels of all four EBP1 orthologs in N. benthamiana were up-regulated by the R. solanacearum infection. The silencing of the four NbEBP1 orthologs suppressed the induction of HR cell death, hin1 expression, and the production of reactive oxygen species. These results suggest that the TOR signaling pathway helps regulate HR cell death along with reactive oxygen species-related signaling in N. benthamiana.

Phosphatidylinositol-phospholipase C4 suppresses the hypersensitive response of Nicotiana benthamiana.

Phospholipid signaling plays an important role in plant immune responses. Here, we isolated two phospholipase C4 (PLC4) orthologs in the Nicotiana benthamiana genome, designated as N. benthamiana PLC4-1 and PLC4-2 (NbPLC4-1 and NbPLC4-2). We created NbPLC4-1- and NbPLC4-2- silenced plants. Induction of the hypersensitive response (HR), including HR cell death and bacterial population reduction, was accelerated in both NbPLC4-1- and NbPLC4-2-silenced plants challenged with N. benthamiana-incompatible Ralstonia solanacearum 8107. The NbPLC4-1- and NbPLC4-2-silenced plants also showed enhanced expression of Nbhin1, a HR marker gene. Expressions of genes for salicylic acid (SA) and jasmonic acid (JA) signaling were drastically increased in NbPLC4-1- and NbPLC4-2-silenced plants by R. solanacearum inoculation. In addition, NbPLC4-1 and NbPLC4-2 silencing triggered reactive oxygen species (ROS) hyper-production. These results suggest that NbPLC4s are closely associated with JA, SA, and ROS signaling and act as negative regulators of the HR in N. benthamiana.

MADS-box protein AGL8 interacts with chromatin-remodelling component SWC4 to activate thermotolerance and environment-dependent immunity in pepper.

Pepper (Capsicum annuum) employs distinct defence responses against Ralstonia solanacearum infection (RSI); however, the mechanisms by which pepper activates these defence responses in a context-dependent manner is unclear. Here we study pepper plants defence response to RSI under room temperature-high humidity (RSRT, 28 °C / 90%) and high temperature-high humidity (RSHT, 37 °C / 90%) conditions, and non-infected plants under high temperature-high humidity (HTHH, 42 °C / 90%) stress. Herein, we found that the MADS-box transcription factor CaAGL8 was up-regulated by HTHH stress and RSRT or RSHT, and its silencing significantly reduced pepper thermotolerance and susceptibility to infection under both room and high temperature-high humidity (RSRT and RSHT). This was coupled with down-regulation of CaSTH2 and CaDEF1 upon RSRT, down-regulation of CaMgst3 and CaPRP1 upon RSHT, and down-regulation of CaHSP24 upon HTHH. In contrast, the ectopic overexpression of CaAGL8 significantly increased the resistance of Nicotiana benthamiana plants to RSRT, RSHT, and HTHH. In addition, CaAGL8 was found to interact with CaSWC4, which acted as a positive regulator of the pepper response to RSRT, RSHT, and HTHH. Silencing of either CaAGL8 or CaSWC4 blocked the hypersensitive response (HR) cell death and context-dependent up-regulation of defence-related genes triggered by the other. Importantly, enrichment of H4K5Ac, H3K9Ac, H3K4me3, and H3K9me2 on the tested defence-related genes was context- and gene-specifically regulated through synergistic interaction between CaSWC4 and CaAGL8. Our results indicate that pepper employs CaAGL8 to modulate chromatin remodelling by interacting with CaSWC4, thereby activating defence responses to RSRT, RSHT, and HTHH.

A rust-fungus Nudix hydrolase effector decaps mRNA invitro and interferes with plant immune pathways.

To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive invitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts invitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage invitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with invitro mRNA-decapping activity capable of interfering with plant immunity.

The Pythium periplocum elicitin PpEli2 confers broad-spectrum disease resistance by triggering a novel receptor-dependent immune pathway in plants.

Elicitins are microbe-associated molecular patterns produced by oomycetes to elicit plant defense. It is still unclear whether elicitins derived from non-pathogenic oomycetes can be used as bioactive molecules for disease control. Here, for the first time we identify and characterize an elicitin named PpEli2 from the soil-borne oomycete Pythium periplocum, which is a non-pathogenic mycoparasite colonizing the root ecosystem of diverse plant species. Perceived by a novel cell surface receptor-like protein, REli, that is conserved in various plants (e.g. tomato, pepper, soybean), PpEli2 can induce hypersensitive response cell death and an immunity response in Nicotiana benthamiana. Meanwhile, PpEli2 enhances the interaction between REli and its co-receptor BAK1. The receptor-dependent immune response triggered by PpEli2 is able to protect various plant species against Phytophthora and fungal infections. Collectively, our work reveals the potential agricultural application of non-pathogenic elicitins and their receptors in conferring broad-spectrum resistance for plant protection.

Plastid methylerythritol phosphate pathway participates in the hypersensitive response-related cell death in Nicotiana benthamiana.

Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.

Rice iron storage protein ferritin 2 (OsFER2) positively regulates ferroptotic cell death and defense responses against Magnaporthe oryzae.

Ferritin is a ubiquitous iron storage protein that regulates iron homeostasis and oxidative stress in plants. Iron plays an important role in ferroptotic cell death response of rice (Oryza sativa) to Magnaporthe oryzae infection. Here, we report that rice ferritin 2, OsFER2, is required for iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death and defense response against the avirulent M. oryzae INA168. The full-length ferritin OsFER2 and its transit peptide were localized to the chloroplast, the most Fe-rich organelle for photosynthesis. This suggests that the transit peptide acts as a signal peptide for the rice ferritin OsFER2 to move into chloroplasts. OsFER2 expression is involved in rice resistance to M. oryzae infection. OsFER2 knock-out in wild-type rice HY did not induce ROS and ferric ion (Fe3+) accumulation, lipid peroxidation and hypersensitive response (HR) cell death, and also downregulated the defense-related genes OsPAL1, OsPR1-b, OsRbohB, OsNADP-ME2-3, OsMEK2 and OsMPK1, and vacuolar membrane transporter OsVIT2 expression. OsFER2 complementation in ΔOsfer2 knock-out mutants restored ROS and iron accumulation and HR cell death phenotypes during infection. The iron chelator deferoxamine, the lipid-ROS scavenger ferrostatin-1, the actin microfilament polymerization inhibitor cytochalasin E and the redox inhibitor diphenyleneiodonium suppressed ROS and iron accumulation and HR cell death in rice leaf sheaths. However, the small-molecule inducer erastin did not trigger iron-dependent ROS accumulation and HR cell death induction in ΔOsfer2 mutants. These combined results suggest that OsFER2 expression positively regulates iron- and ROS-dependent ferroptotic cell death and defense response in rice-M. oryzae interactions.

The NIN-Like Protein OsNLP2 Negatively Regulates Ferroptotic Cell Death and Immune Responses to Magnaporthe oryzae in Rice.

Nodule inception (NIN)-like proteins (NLPs) have a central role in nitrate signaling to mediate plant growth and development. Here, we report that OsNLP2 negatively regulates ferroptotic cell death and immune responses in rice during Magnaporthe oryzae infection. OsNLP2 was localized to the plant cell nucleus, suggesting that it acts as a transcription factor. OsNLP2 expression was involved in susceptible disease development. ΔOsnlp2 knockout mutants exhibited reactive oxygen species (ROS) and iron-dependent ferroptotic hypersensitive response (HR) cell death in response to M. oryzae. Treatments with the iron chelator deferoxamine, lipid-ROS scavenger ferrostatin-1, actin polymerization inhibitor cytochalasin A, and NADPH oxidase inhibitor diphenyleneiodonium suppressed the accumulation of ROS and ferric ions, lipid peroxidation, and HR cell death, which ultimately led to successful M. oryzae colonization in ΔOsnlp2 mutants. The loss-of-function of OsNLP2 triggered the expression of defense-related genes including OsPBZ1, OsPIP-3A, OsWRKY104, and OsRbohB in ΔOsnlp2 mutants. ΔOsnlp2 mutants exhibited broad-spectrum, nonspecific resistance to diverse M. oryzae strains. These combined results suggest that OsNLP2 acts as a negative regulator of ferroptotic HR cell death and defense responses in rice, and may be a valuable gene source for molecular breeding of rice with broad-spectrum resistance to blast disease.

TuRLK1, a leucine-rich repeat receptor-like kinase, is indispensable for stripe rust resistance of YrU1 and confers broad resistance to multiple pathogens.

BackgroundYrU1 is a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) protein (NLR), with additional ankyrin-repeat and WRKY domains and confers effective resistance to stripe rust fungus Puccinia striiformis f. sp. Tritici (Pst). YrU1 was positionally cloned in the progenitor species of the A genome of bread wheat, Tricicum urartu, recently. However, the molecular mechanism and components involved in YrU1-mediated resistance are not clear.ResultsIn this study, we found that the transcript level of TuRLK1, which encodes a novel leucine-rich repeat receptor-like kinase, was up-regulated after inoculation with Pst in the presence of YrU1, through RNA-seq analysis in T. urartu accession PI428309. TuRLK1 contained only a small number of LRR motifs, and was localized in the plasma-membrane. Transient expression of TuRLK1 induced hypersensitive cell death response in N. benthamiana leaves. Silencing of TuRLK1, using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system in PI428309 that contains YrU1, compromised the resistance against stripe rust caused by Pst CY33, indicating that TuRLK1 was required for YrU1-activated plant immunity. Furthermore, overexpression of TuRLK1 could enhance powdery mildew resistance in bread wheat and Arabidopsis thaliana after inoculating with the corresponding pathogens.ConclusionsOur study indicates that TuRLK1 is required for immune response mediated by the unique NLR protein YrU1, and likely plays an important role in disease resistance to other pathogens.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-022-03679-6.

Optimization of immune receptor-related hypersensitive cell death response assay using agrobacterium-mediated transient expression in tobacco plants

BackgroundThe study of the regulatory mechanisms of evolutionarily conserved Nucleotide-binding leucine-rich repeat (NLR) resistance (R) proteins in animals and plants is of increasing importance due to understanding basic immunity and the value of various crop engineering applications of NLR immune receptors. The importance of temperature is also emerging when applying NLR to crops responding to global climate change. In particular, studies of pathogen effector recognition and autoimmune activity of NLRs in plants can quickly and easily determine their function in tobacco using agro-mediated transient assay. However, there are conditions that should not be overlooked in these cell death-related assays in tobacco.ResultsEnvironmental conditions play an important role in the immune response of plants. The system used in this study was to establish conditions for optimal hypertensive response (HR) cell death analysis by using the paired NLR RPS4/RRS1 autoimmune and AvrRps4 effector recognition system. The most suitable greenhouse temperature for growing plants was fixed at 22 °C. In this study, RPS4/RRS1-mediated autoimmune activity, RPS4 TIR domain-dependent cell death, and RPS4/RRS1-mediated HR cell death upon AvrRps4 perception significantly inhibited under conditions of 65% humidity. The HR is strongly activated when the humidity is below 10%. Besides, the leaf position of tobacco is important for HR cell death. Position #4 of the leaf from the top in 4–5 weeks old tobacco plants showed the most effective HR cell death.ConclusionsAs whole genome sequencing (WGS) or resistance gene enrichment sequencing (RenSeq) of various crops continues, different types of NLRs and their functions will be studied. At this time, if we optimize the conditions for evaluating NLR-mediated HR cell death, it will help to more accurately identify the function of NLRs. In addition, it will be possible to contribute to crop development in response to global climate change through NLR engineering.

Evolutionary Diversity and Function of Metacaspases in Plants: Similar to but Not Caspases.

Caspase is a well-studied metazoan protease involved in programmed cell death and immunity in animals. Obviously, homologues of caspases with evolutionarily similar sequences and functions should exist in plants, and yet, they do not exist in plants. Plants contain structural homologues of caspases called metacaspases, which differ from animal caspases in a rather distinct way. Metacaspases, a family of cysteine proteases, play critical roles in programmed cell death during plant development and defense responses. Plant metacaspases are further subdivided into types I, II, and III. In the type I Arabidopsis MCs, AtMC1 and AtMC2 have similar structures, but antagonistically regulate hypersensitive response cell death upon immune receptor activation. This regulatory action is similar to caspase-1 inhibition by caspase-12 in animals. However, so far very little is known about the biological function of the other plant metacaspases. From the increased availability of genomic data, the number of metacaspases in the genomes of various plant species varies from 1 in green algae to 15 in Glycine max. It is implied that the functions of plant metacaspases will vary due to these diverse evolutions. This review is presented to comparatively analyze the evolution and function of plant metacaspases compared to caspases.

A prophage-encoded nonclassical secretory protein of "Candidatus Liberibacter asiaticus" induces a strong immune response in Nicotiana benthamiana and citrus.

Huanglongbing (HLB), associated with “Candidatus Liberibacter asiaticus” (CLas), is a globally devastating plant disease. The highly reduced genome of CLas encodes a number of secretory proteins. The conserved prophage‐encoded protein AGH17470 is herein identified as a nonclassical secretory protein. We confirmed that the N‐terminal and C‐terminal sequences jointly determine the secretion of AGH17470. The transient expression of AGH17470 protein in Nicotiana benthamiana caused hypersensitive response (HR) cell death in infiltrated leaves and systemically infected leaves as well as the dwarfing of the entire plant, suggesting that AGH17470 is involved in the plant immune response, growth, and development. Overexpression of AGH17470 in N. benthamiana and citrus plants up‐regulated the transcription of pathogenesis‐related and salicylic acid (SA)‐signalling pathway genes and promoted SA accumulation. Furthermore, transient expression of AGH17470 enhanced the resistance of sweet orange to Xanthomonas citri subsp. citri. To our knowledge, AGH17470 is the first prophage‐encoded secretory protein demonstrated to elicit an HR and induce a strong plant immune response. The findings have increased our understanding of prophage‐encoded secretory protein genes, and the results provide clues as to the plant defence response against CLas.

Functional analysis of hot pepper ethylene responsive factor 1A in plant defense

ABSTRACT Ethylene-responsive factors play important roles in the biotic and abiotic stresses. Only some ERF genes from Capsicum annuum have been characterized. In the study, the CaERF1A gene is characterized in response to biotic stress. CaERF1A transcripts were induced by various plant defense-related hormone treatments. Knockdown of CaERF1A in hot pepper plants are negatively affected Tobacco mosaic virus-P0-mediated hypersensitive response cell death, resulting in reduced gene expression of pathogenesis-related genes and ethylene and jasmonic acid synthesis-related gene. Overexpressing CaERF1A transgenic plants show enhanced resistance to fungal pathogen via regulating ethylene and jasmonic acid synthesis-related gene expression. Thus, CaERF1A is a positive regulator of plant defense by modulating ethylene and jasmonic acid synthesis-related gene expressions.