Lesion Mimic Phenotype Research Articles

Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.

OsNSUN2-Mediated 5-Methylcytosine mRNA Modification Enhances Rice Adaptation to High Temperature.

Extreme weather events can cause heat stress that decreases crop production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in the thermoresponse in plants. However, how RNA modifications contribute to temperature stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as an RNA 5-methylcytosine (m5C) methyltransferase in rice. osnsun2 mutant displayed severe temperature- and light-dependent lesion-mimic phenotypes and heat-stress hypersensitivity. Heat stress enhanced the OsNSUN2-dependent m5C modification of mRNAs involved in photosynthesis and detoxification systems, such as β-OsLCY, OsHO2, OsPAL1, and OsGLYI4, which increased protein synthesis. Furthermore, the photosystem of osnsun2 mutant was vulnerable to high ambient temperature and failed to undergo repair under tolerable heat stress. Thus, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.

Characterization and Genetic Mapping of White-Spotted Leaf (wspl) Mutant in Rice

Spotted leaf mutants which produce necrotic lesions spontaneously are important sources to study programmed cell death in plant defense responses. A novel white-spotted leaf (wspl) mutant was induced from Ilpum, Korean japonica rice cultivar by the treatment of ethyl methane sulfonate (EMS). The phenotype of wspl mutant differed from that of other spotted leaf mutants in that not only brown spots but also white lesion mimic spots were observed on the tip of the leaves from the vegetative stage. Strong nitro blue tetrazolium (NBT) and 3, 3′-diaminobenzidine (DAB) staining were observed on the older leaf of wspl mutant in microscopic reactive oxygen species (ROS) assay, and the chlorophyll content of wspl mutant maintained longer than wild-type in the old leaves. Genetic analysis revealed that the wspl mutant trait was controlled by a single recessive gene and the locus of wspl gene was mapped on the long arm of chromosome 5 between the flanking markers S05100 and S05112 (4.1 Mb). Through the combination of the genetic mapping and SNP analysis, two candidate genes for white-spotted leaf were identified in the genic region. A novel phenotype of white-spotted leaf mutant has not yet been reported, thus further study of the wspl mutant will contribute to understanding of the molecular mechanisms involved in lesion mimic phenotype in rice.

Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

BackgroundVerticillium wilt, caused by Verticillium dahliae, is called a “cancer” disease of cotton. The discovery and identification of defense-related genes is essential for the breeding of Verticillium wilt-resistant varieties. In previous research we identified some possible broad-spectrum resistance genes. Here, we report a tryptophan synthesis-related gene GbTRP1 and its functional analysis in relation to the resistance of cotton to V. dahliae.ResultsExpression analysis shows that GbTRP1 is suppressed at 1 h and 6 h post V. dahliae infection, but activated at 12 h and 24 h, and the expression of GbTRP1 is highly induced by treatment with salicylic acid and jasmonic acid. Sub-cellular localization studies show that GbTRP1 is localized in the chloroplast. Suppression of GbTRP1 expression leads to lesion-mimic phenotypes and activates the immune response in cotton by showing enhanced resistance to V. dahliae and B. cinerea. Metabolomic analysis shows that anthranilic compounds significantly accumulated in GbTRP1-silenced plants, and these metabolites can inhibit the growth of V. dahliae and B. cinerea in vitro.ConclusionsOur results show that suppression of GbTRP1 expression dramatically activates the immune response and increases resistance of cotton to V. dahliae and B. cinerea, possibly due to the accumulation of anthranilate compounds. This study not only provides genetic resources for disease resistance breeding, but also may provide a basis for new chemical control methods for combatting of fungal disease in cotton.

Characterization and Rapid Gene-Mapping of Leaf Lesion Mimic Phenotype of spl-1 Mutant in Soybean (Glycine max (L.) Merr.).

In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (spl-1) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (lm1 locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5′ or 3′ UTR. Hence, the data of the present study are in favor of Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.

Identification and expression analysis of the arbuscular mycorrhiza-inducible Rieske non-heme oxygenase Ptc52 gene from tomato

Arbuscular mycorrhizal (AM) formation enhances plant growth and fitness through improved uptake of water and mineral nutrients in exchange for carbon compounds to the AM fungus. The fungal structure for the reciprocal exchange of nutrients in the symbiosis is the arbuscule, and defence genes expressed in cells containing arbuscules could play a role in the control of hyphal spread and arbuscule formation in the root. We characterized and analyzed the Ptc52 gene from tomato (SlPtc52), a member of the gene family of non-heme oxygenases, whose function has been related to the lethal leaf spot 1 (Lls1) lesion mimic phenotype in plants which is sometimes associated with enhanced disease resistance. Sequence analysis of the SlPTC52 protein revealed conserved typical motifs from non-heme oxygenases, including a Rieske [2Fe-2S] motif, a mononuclear non-heme iron-binding motif and a C-terminal CxxC motif. The level of transcript accumulation was low in stem, flower and green fruits, and high in leaves. Although SlPtc52 expression was perceptible at low levels in roots, its expression increased concomitantly with AM fungus root colonization. Tomato non-mycorrhizal hairy roots expressing the GUS protein under the control of promoter SlPtc52 exhibited GUS activity in the endodermis, the apical meristem of the root tip and in the lateral root primordium. AM fungal colonization also resulted in intensive GUS activity that clearly corresponds to cortical cells containing arbuscules. SlPtc52 gene silencing led to a delay in root colonization and a decrease in arbuscular abundance, suggesting that SlPTC52 plays a regulatory role during AM symbiosis.

Disruption of gene SPL35, encoding a novel CUE domain-containing protein, leads to cell death and enhanced disease response in rice.

SummaryLesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H2O2, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.

Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

Mutation in a putative glycosyltransferase-like gene causes programmed cell death and early leaf senescence in rice

Leaf senescence is a genetically regulated, highly complex and ordered process. Although it has been extensively studied, the mechanism of leaf senescence is not well understood. In this study, we isolated a rice mutant, designated as premature senescence leaf (psl), which exhibits early senescence and spontaneous lesion mimic phenotype after flowering. The psl mutant displays programmed cell death with elevated accumulation of reactive oxygen species (ROS). Molecular and genetic analyses revealed that the phenotypes were caused by a phenylalanine deletion in the OsPSL (LOC_Os12g42420) that encode a putative core 2/I branching beta-1,6-N-acetylglucosaminyl transferase predicted to be involved in protein glycosylation modification. OsPSL mRNA levels increased as senescence progressed, with maximum accumulation of transcripts at late senescence stages in WT plants. Moreover, remarkedly down-regulated transcriptional levels of O-linked N-acetylglucosamine (O-GlcNAc) transferases (OGTs) genes were observed in psl mutant, supporting the occurrence of impaired O-glycosylation modification. Proteomic analysis showed that ethylene-related metabolic enzymes including S-adenosyl methionine (SAM) synthetase (SAMS) were significantly upregulated in the psl mutant compared with WT. Consistent with the proteomic results, ethylene concentration is higher in psl mutant than in wild-type plants, and transcript levels of ethylene synthesis and signal transduction genes were induced in psl mutant. The early leaf senescence of psl can be partially rescued by ethylene biosynthesis inhibitor aminoethoxyvinylglycine treatment. These results highlight the importance of protein O-glycosylation in PCD and leaf senescence, and suggest a possible role of OsPSL in ethylene signaling.

OsACL-A2 negatively regulates cell death and disease resistance in rice.

Summary ATP‐citrate lyases (ACL) play critical roles in tumour cell propagation, foetal development and growth, and histone acetylation in human and animals. Here, we report a novel function of ACL in cell death‐mediated pathogen defence responses in rice. Using ethyl methanesulphonate (EMS) mutagenesis and map‐based cloning, we identified an Oryza sativa ACL‐A2 mutant allele, termed spotted leaf 30‐1 (spl30‐1), in which an A‐to‐T transversion converts an Asn at position 343 to a Tyr (N343Y), causing a recessive mutation that led to a lesion mimic phenotype. Compared to wild‐type plants, spl30‐1 significantly reduces ACL enzymatic activity, accumulates high reactive oxygen species and increases degradation rate of nuclear deoxyribonucleic acids. CRISPR/Cas9‐mediated insertion/deletion mutation analysis and complementation assay confirmed that the phenotype of spl30‐1 resulted from the defective function of OsACL‐A2 protein. We further biochemically identified that the N343Y mutation caused a significant degradation of SPL30N343Y in a ubiquitin‐26S proteasome system (UPS)‐dependent manner without alteration in transcripts of OsACL‐A2 in spl30‐1. Transcriptome analysis identified a number of up‐regulated genes associated with pathogen defence responses in recessive mutants of OsACL‐A2, implying its role in innate immunity. Suppressor mutant screen suggested that OsSL, which encodes a P450 monooxygenase protein, acted as a downstream key regulator in spl30‐1‐mediated pathogen defence responses. Taken together, our study discovered a novel role of OsACL‐A2 in negatively regulating innate immune responses in rice.

Proteome analysis and phenotypic characterization of the lesion mimic mutant bspl in barley

Characterization and molecular mechanism dissection for lesion-mimic mutation can help to gain an in-depth insight into regulatory mechanisms of programmed cell death in plants. In present study, the barley lesion mimic mutant bspl (barley spotted 1eaf) was used to investigate the formation of the bspl lesion mimic phenotype in barley using proteomics and quantitative real-time (qRT)-PCR techniques. The experiment showed that the formation of the bspl phenotype was directly related to light. Cell necrosis spontaneously formed in multiple parts of the leaf, and H2O2 accumulated continuously along with the formation of lesions. A total of 31 differentially expressed protein spots were identified using two-dimensional electrophoresis and mass spectrometry analysis of the expressed proteins between the bspl and the wild-type. These differentially expressed proteins are involved in different biological processes, mainly related to defense, photosynthesis, nitrogen acid metabolism, and carbohydrate metabolism. The expression of 15 genes was analyzed by qRT-PCR, and the results showed that the transcription level was basically consistent with the change in the protein level. The decreased expression of ATPase β subunit, cytochrome B6-F complex, photosystem I reaction center subunit VI, and other proteins may lead to the blockage of electron transport during photosynthesis, leading to cell death. Moreover, the overexpression of chitinase, thaumatin-like protein, PR-1a and other proteins, and the increased expression of NADPH-thioredoxin reductase and dehydroascorbate reductase may increase the disease resistance and stress resistance of bspl. This study will provide a new insight into the molecular mechanism underlying bspl-induced cell death in plants.

Identification of a Novel Semi-Dominant Spotted-Leaf Mutant with Enhanced Resistance to Xanthomonas oryzae pv. oryzae in Rice.

Many spotted-leaf mutants show enhanced disease resistance to multiple pathogen attacks; however, the mechanisms are largely unknown. Here, we reported a novel semi-dominant spotted-leaf mutant 24 (spl24) obtained from an ethyl methane sulfonate (EMS)-induced IR64 mutant bank. spl24 developed tiny brown lesions on the leaf tip and spread down gradually to the leaf base as well as the sheath at the early heading stage. The performances of major agronomic traits such as the plant height, panicle length, number of panicles/plant, and 1000-grain weight were significantly altered in spl24 when compared to the wild-type IR64. Furthermore, spl24 exhibited a premature senescing phenotype with degeneration of nuclear acids, significantly reduced soluble protein content, increased level of malonaldehyde (MDA), and lowered activities of reactive oxygen species (ROS) scavenging enzymes. Disease evaluation indicated that spl24 showed enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial leaf blight in rice, with elevated expression of pathogenesis-related genes, salicylic acid (SA) signaling pathway-associated genes revealed by real-time quantitative PCR and high-throughput RNA sequencing analysis. Genetic analysis and gene mapping indicated that the lesion mimic phenotype was controlled by a novel semi-dominant nuclear gene. The mutation, tentatively termed as OsSPL24, was in a 110 kb region flanked by markers Indel-33 and Indel-12 in chromosome 11. Together, our data suggest that spl24 is a novel lesion mimic mutant with enhanced innate immunity and would facilitate the isolation and functional characterization of the target gene.

Arabidopsis UBC13 differentially regulates two programmed cell death pathways in responses to pathogen and low-temperature stress.

UBC13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity remain to be defined. Here we used genetic and pathological methods to evaluate roles of Arabidopsis UBC13 in response to pathogens and environmental stresses. Loss of UBC13 failed to activate the expression of numerous cold-responsive genes and resulted in hypersensitivity to low-temperature stress, indicating that UBC13 is involved in plant response to low-temperature stress. Furthermore, the ubc13 mutant displayed low-temperature-induced and salicylic acid-dependent lesion mimic phenotypes. Unlike typical lesion mimic mutants, ubc13 did not enhance disease resistance against virulent bacterial and fungal pathogens, but diminished hypersensitive response and compromised effector-triggered immunity against avirulent bacterial pathogens. UBC13 differently regulates two types of programmed cell death in response to low temperature and pathogen. The lesion mimic phenotype in the ubc13 mutant is partially dependent on SNC1. UBC13 interacts with an F-box protein CPR1 that regulates the homeostasis of SNC1. However, the SNC1 protein level was not altered in the ubc13 mutant, implying that UBC13 is not involved in CPR1-regulated SNC1 protein degradation. Taken together, our results revealed that UBC13 is a key regulator in plant response to low temperature and pathogens.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Modulation of Plant Salicylic Acid-Associated Immune Responses via Glycosylation of Dihydroxybenzoic Acids.

Salicylic acid (SA) plays a crucial role in plant innate immunity. The deployment of SA-associated immune responses is primarily affected by SA concentration, which is determined by a balance between SA biosynthesis and catabolism. However, the mechanisms regulating SA homeostasis are poorly understood. In this study, we characterized a unique UDP-glycosyltransferase, UGT76D1, which plays an important role in SA homeostasis and associated immune responses in Arabidopsis (Arabidopsis thaliana). Expression of UGT76D1 was induced by treatment with both the pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 and SA. Overexpression of UGT76D1 resulted in high SA accumulation, significant up-regulation of pathogen-related genes, and a hypersensitive response (HR)-like lesion mimic phenotype. This HR-like phenotype was not observed following UGT76D1 overexpression in SA-deficient NahG transgenic or sid2 plants, suggesting that the phenotype is SA dependent. Biochemical assays showed that UGT76D1 glycosylated 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), the major catabolic forms of SA, to their Glc and Xyl conjugates in vitro and in vivo. Moreover, in a mutant background blocked in the formation of 2,3-DHBA and 2,5-DHBA, UGT76D1 overexpression did not cause a HR-like lesion mimic phenotype. Following infection with Pst DC3000, UGT76D1 knockout mutants displayed a delayed immune response, with reduced levels of DHBA glycosides and SA, and down-regulated SA synthase expression. By contrast, UGT76D1 overexpression lines showed an enhanced immune response and increased SA biosynthesis before and after pathogen infection. Thus, we propose that UGT76D1 plays an important role in SA homeostasis and plant immune responses by facilitating glycosylation of dihydroxybenzoic acids.

Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice.

The exocyst, an evolutionarily conserved octameric protein complex involved in exocytosis, has been reported to be involved in diverse aspects of morphogenesis in Arabidopsis. However, the molecular functions of such exocytotic molecules in rice are poorly understood. Here, we examined the molecular function of OsSEC3A, an important subunit of the exocyst complex in rice. The OsSEC3A gene is expressed in various organs, and OsSEC3A has the potential ability to participate in the exocyst complex by interacting with several other exocyst subunits. Disruption of OsSEC3A by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) caused dwarf stature and a lesion-mimic phenotype. The Ossec3a mutant exhibited enhanced defense responses, as shown by up-regulated transcript levels of pathogenesis- and salicylic acid synthesis-related genes, increased levels of salicylic acid, and enhanced resistance to the fungal pathogen Magnaporthe oryzae. Subcellular localization analysis demonstrated that OsSEC3A has a punctate distribution with the plasma membrane. In addition, OsSEC3A interacted with rice SNAP25-type t-SNARE protein OsSNAP32, which is involved in rice blast resistance, via the C-terminus and bound to phosphatidylinositol lipids, particularly phosphatidylinositol-3-phosphate, through its N-terminus. These findings uncover the novel function of rice exocyst subunit SEC3 in defense responses.

Characterization and Identification of a woody lesion mimic mutant lmd, showing defence response and resistance to Alternaria alternate in birch

Lesion mimic mutants (LMM) usually show spontaneous cell death and enhanced defence responses similar to hypersensitive response (HR) in plants. Many LMM have been reported in rice, wheat, maize, barley, Arabidopsis, etc., but little was reported in xylophyta. BpGH3.5 is an early auxin-response factor which regulates root elongation in birch. Here, we found a T-DNA insertion mutant in a BpGH3.5 transgenic line named lmd showing typical LMM characters and early leaf senescence in Betula platyphylla × B. pendula. lmd showed H2O2 accumulation, increased SA level and enhanced resistance to Alternaria alternate, compared with oe21 (another BpGH3.5 transgenic line) and NT (non-transgenic line). Cellular structure observation showed that programmed cell death occurred in lmd leaves. Stereomicroscope observation and Evans’ blue staining indicated that lmd is a member of initiation class of LMM. Transcriptome analysis indicated that defence response-related pathways were enriched. Southern-blot indicated that there were two insertion sites in lmd genome. Genome re-sequencing and thermal asymmetric interlaced PCR (TAIL-PCR) confirmed the two insertion sites, one of which is a T-DNA insertion in the promoter of BpEIL1 that may account for the lesion mimic phenotype. This study will benefit future research on programmed cell death, HR and disease resistance in woody plants.

An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens

Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid-binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant vad1-1 has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, associated with defense gene expression and increased resistance to Pseudomonas strains. To test the potential of ectopic expression of VAD1 to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure-function analysis of VAD1 by transient over-expression in Nicotiana benthamiana and by complementation of the mutant vad1-1. We found that (i) overexpression of VAD1 controls negatively the HR cell death and defense expression either transiently in Nicotiana benthamania or in Arabidopsis plants in response to avirulent strains of Pseudomonas syringae, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death.

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice.

Cullin3-based RING E3 ubiquitin ligases (CRL3), composed of Cullin3 (CUL3), RBX1, and BTB proteins, are involved in plant immunity, but the function of CUL3 in the process is largely unknown. Here, we show that rice (Oryza sativa) OsCUL3a is important for the regulation of cell death and immunity. The rice lesion mimic mutant oscul3a displays a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in pathogenesis-related gene expression as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. We cloned the OsCUL3a gene via a map-based strategy and found that the lesion mimic phenotype of oscul3a is associated with the early termination of OsCUL3a protein. Interaction assays showed that OsCUL3a interacts with both OsRBX1a and OsRBX1b to form a multisubunit CRL in rice. Strikingly, OsCUL3a interacts with and degrades OsNPR1, which acts as a positive regulator of cell death in rice. Accumulation of OsNPR1 protein is greater in the oscul3a mutant than in the wild type. Furthermore, the oscul3a osnpr1 double mutant does not exhibit the lesion mimic phenotype of the oscul3a mutant. Our data demonstrate that OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice.