Lesion Mimic Phenotype Research Articles

Ectopic Expression of the Executor-Type R Gene Paralog Xa27B in Rice Leads to Spontaneous Lesions and Enhanced Disease Resistance.

Plant disease resistance (R) gene-mediated effector-triggered immunity (ETI) is usually associated with hypersensitive response (HR) and provides robust and race-specific disease resistance against pathogenic infection. The activation of ETI and HR in plants is strictly regulated, and improper activation will lead to cell death. Xa27 is an executor-type R gene in rice induced by the TAL effector AvrXa27 and confers disease resistance to Xanthomonas oryzae pv. oryzae (Xoo). Here we reported the characterization of a transgenic line with lesion mimic phenotype, designated as Spotted leaf and resistance 1 (Slr1), which was derived from rice transformation with a genomic subclone located 5,125 bp downstream of the Xa27 gene. Slr1 develops spontaneous lesions on its leaves caused by cell death and confers disease resistance to both Xoo and Xanthomonas oryzae pv. oryzicola. Further investigation revealed that the Slr1 phenotype resulted from the ectopic expression of an Xa27 paralog gene, designated as Xa27B, in the inserted DNA fragment at the Slr1 locus driven by a truncated CaMV35Sx2 promoter in reverse orientation. Disease evaluation of IRBB27, IR24, and Xa27B mutants with Xoo strains expressing dTALE-Xa27B confirmed that Xa27B is a functional executor-type R gene. The functional XA27B-GFP protein was localized to the endoplasmic reticulum and apoplast. The identification of Xa27B as a new functional executor-type R gene provides additional genetic resources for studying the mechanism of executor-type R protein-mediated ETI and developing enhanced and broad-spectrum disease resistance to Xoo through promoter engineering. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role inRYMVsusceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Effect of RNAi suppression of the gossypol pathway on resistance to necrotrophic fungal seedling disease pathogens in cotton

We previously demonstrated that blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the hemibiotrophic Fusarium wilt pathogens. The enhanced resistance was attributed to the induction of a linked lacinilene defense pathway. Others reported that RNAi suppression of the same gene, which was named as SILENCE-INDUCED STEM NECROSIS (SSN), caused spontaneous hypersensitive response-like cell death resulting in a lesion mimic phenotype and increased resistance to hemibiotrophic Verticillium wilt pathogens. We observed no lesion formation on hypocotyls when the CYP82D RNAi plants were grown in pasteurized soil at warm temperatures that are unfavorable to seedling disease development. Therefore, RNAi suppression of CYP82D is not associated with spontaneous hypersensitive-like cell death. When grown in unpasteurized soil at cool temperatures that are favorable for seedling disease development, more than 87% of the CYP82D RNAi plants developed necrotic hypocotyl lesions, compared to 3% of the wild type plants that developed lesions. Furthermore, at least 83% of the necrotic CYP82D RNAi plants harbored at least one of the isolates of Fusarium spp, Rhizopus spp., or Aspergillus spp. that are often implicated in the seedling diseases of cotton. Pathogenicity assays with the seedling disease pathogen Rhizoctonia solani AG4 confirmed that blocking the gossypol defense pathway by CYP82D RNAi increased host susceptibility to the necrotrophic pathogen. The concentrations of induced lacinilene products detected in the roots and hypocotyls of RNAi plants when treated with Rhizoctonia were far lower than the concentrations seen previously in these tissues induced by Fusarium wilt pathogens. Thus, the increased susceptibility of the RNAi plants to Rhizoctonia was probably due to the combined effect of low concentrations of lacinilenes and the blockage of the gossypol pathway by the RNAi. Developing cotton host resistance to wilt diseases without compromising resistance to seedling disease pathogens requires further understanding of the interaction between signaling pathways involved in responses to hemibiotrophs and necrotrophs.

Lesion mimic mutant 8 balances disease resistance and growth in rice.

Lesion-mimic mutants (LMM) spontaneously produce necrotic spots, a process not affected by environmental stress or pathogen infection. In this study, we identified a LMM, lesion mimic mutant 8 (lmm8) in rice (Oryza sativa). The lmm8 mutant produces brown and off-white lesions on its leaves during the second- and third-leaf stages. The lesion mimic phenotype of the lmm8 mutant was enhanced by light. At the mature stage, lmm8 mutant are shorter and exhibit inferior agronomic traits than the wild type. Contents of photosynthetic pigments and chloroplast fluorescence were significantly reduced in lmm8 leaves, along with increased production of reactive oxygen species and programmed cell death compared to the wild type. The mutated gene was identified as LMM8 (LOC_Os01g18320) by map-based cloning. A point mutation occurred in LMM8, causing a Leu to Arg mutation of the 146th amino acid of LMM8. It is an allele of SPRL1, encoding a protoporphyrinogen IX oxidase (PPOX) located in chloroplasts and involved in the biosynthesis of tetrapyrrole in chloroplasts. The lmm8 mutant showed enhanced resistance and broad-spectrum resistance. Together, our results demonstrate the importance of rice LMM8 protein in defense responses and plant growth in rice, and provides theoretical support for resistance breeding to improve rice yield.

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

MEDIATOR SUBUNIT 16 negatively regulates rice immunity by modulating PATHOGENESIS RELATED 3 activity.

Lesion mimic mutants (LMMs) are valuable genetic resources for unraveling plant defense responses including programmed cell death. Here, we identified a rice (Oryza sativa) LMM, spotted leaf 38 (spl38), and demonstrated that spl38 is essential for the formation of hypersensitive response-like lesions and innate immunity. Map-based cloning revealed that SPL38 encodes MEDIATOR SUBUNIT 16 (OsMED16). The spl38 mutant showed enhanced resistance to rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) and exhibited delayed flowering, while OsMED16-overexpressing plants showed increased rice susceptibility to M. oryzae. The OsMED16-edited rice lines were phenotypically similar to the spl38 mutant but were extremely weak, exhibited growth retardation, and eventually died. The C-terminus of OsMED16 showed interaction with the positive immune regulator PATHOGENESIS RELATED 3 (OsPR3), resulting in the competitive repression of its chitinase and chitin-binding activities. Furthermore, the ospr3 osmed16 double mutants did not exhibit the lesion mimic phenotype of the spl38 mutant. Strikingly, OsMED16 exhibited an opposite function in plant defense relative to that of Arabidopsis (Arabidopsis thaliana) AtMED16, most likely because of 2 amino acid substitutions between the monocot and dicot MED16s tested. Collectively, our findings suggest that OsMED16 negatively regulates cell death and immunity in rice, probably via the OsPR3-mediated chitin signaling pathway.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice

ABSTRACT Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D underlies lesion-mimic phenotype in dark-treated Arabidopsis pheide a oxygenase mutant leaves.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) encoding a plasma membrane NADPH oxidase is responsible for the lesion-mimic phenotype in detached Arabidopsis leaves with mutation of PHEIDE a OXYGENASE during extended darkness. Chlorophyll degradation is an indispensable process in leaf senescence, either age-dependent or dark-induced. Besides higher chlorophyll retention, a lesion-mimic phenotype (abbreviated as LMP afterwards) was exhibited in Arabidopsis leaves with mutation of PHEIDE a OXYGENASE (PaO) involved in chlorophyll degradation during dark incubation, but the associated mechanism remains elusive. We found that dark-treated pao leaves showed higher membrane damage and H2O2 accumulation, while scavenging H2O2 by its chemical scavenger diminished LMP. RBOHD which encodes NADPH oxidase was strikingly up-regulated in pao leaves during dark treatment. Chemical inhibition of NADPH oxidase or mutation of RBOHD in pao leaves suppressed LMP. Thus, our study suggests that up-regulated RBOHD transcription is responsible for the formation of LMP in dark-treated pao leaves and there may be a retrograde signaling pathway mediating upregulation of RBOHD which remains to be elucidated.

OsLMP1, Encoding a Deubiquitinase, Regulates the Immune Response in Rice.

Lesion mimic mutants have become an effective material for understanding plant-microbe interactions and the immune mechanism in plants. Although many mechanisms responsible for the lesion mimic phenotype have been clarified in plants, the mechanism by which lesion mimic is regulated by posttranslational modification remained largely elusive, especially in rice. In this study, a mutant with the lesion mimic phenotype was obtained and named lmp1-1. Physiological measurements and quantitative real-time PCR analysis showed that the defense response was activated in the mutants. Transcriptome analysis showed that the phenylalanine ammonia lyase (PAL) pathway was activated in the mutant, causing the accumulation of salicylic acid (SA). The results of mapping based cloning showed that OsLMP1 encodes a deubiquitinase. OsLMP1 can cleave ubiquitination precursors. Furthermore, OsLMP1 epigenetically modifies SA synthetic pathway genes by deubiquitinating H2B and regulates the immune response in rice. In summary, this study deepens our understanding of the function of OsLMP1 in the plant immune response and provides further insight into the relationship between plants and pathogenic microorganisms.

Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.

Summary Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane‐related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes.CRISPR/Cas9‐mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs.Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono‐oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted.These results reveal directional cross‐cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein, regulates light-dependent cell death and defence responses in rice.

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross‐talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map‐based cloning, a short‐chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7‐6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light‐dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity‐related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000‐grain weight, grain weight per plant, panicle length, and plant height. SDR7‐6 is a new lesion mimic gene that encodes a short‐chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7‐6 led to cell death and had adverse effects on multiple agricultural characters. SDR7‐6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.

OsNBL3, a mitochondrion-localized pentatricopeptide repeat protein, is involved in splicing nad5 intron 4 and its disruption causes lesion mimic phenotype with enhanced resistance to biotic and abiotic stresses.

SummaryLesion mimic mutants are used to elucidate mechanisms controlling plant responses to pathogen attacks and environmental stresses. Although dozens of genes had been functionally demonstrated to be involved in lesion mimic phenotype in several plant species, the molecular mechanisms underlying the hypersensitive response are largely unknown. Here, a rice (Oryza sativa) lesion mimic mutant natural blight leaf 3 (nbl3) was identified from T‐DNA insertion lines. The causative gene, OsNBL3, encodes a mitochondrion‐localized pentatricopeptide repeat (PPR) protein. The nbl3 mutant exhibited spontaneous cell death response and H2O2 accumulation, and displayed enhanced resistance to the fungal and bacterial pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. This resistance was consistent with the up‐regulation of several defence‐related genes; thus, defence responses were induced in nbl3. RNA interference lines of OsNBL3 exhibited enhanced disease resistance similar to that of nbl3, while the disease resistance in overexpression lines did not differ from that of the wild type. In addition, nbl3 displayed improved tolerance to salt, accompanied by up‐regulation of several salt‐associated marker genes. OsNBL3 was found to mainly participate in the splicing of mitochondrial gene nad5 intron 4. Disruption of OsNBL3 leads to the reduction in complex I activity, the elevation of alternative respiratory pathways and the destruction of mitochondrial morphology. Overall, the results demonstrated that the PPR protein‐coding gene OsNBL3 is essential for mitochondrial development and functions, and its disruption causes the lesion mimic phenotype and enhances disease resistance and tolerance to salt in rice.

A teosinte-derived allele of a MYB transcription repressor confers multiple disease resistance in maize

Natural alleles that control multiple disease resistance (MDR) are valuable for crop breeding. However, only one MDR gene has been cloned in maize, and the molecular mechanisms of MDR remain unclear in maize. In this study, through map-based cloning we cloned a teosinte-derived allele of a resistance gene, Mexicana lesion mimic 1 (ZmMM1), which causes a lesion mimic phenotype and confers resistance to northern leaf blight (NLB), gray leaf spot (GLS), and southern corn rust (SCR) in maize. Strong MDR conferred by the teosinte allele is linked with polymorphisms in the 3′ untranslated region of ZmMM1 that cause increased accumulation of ZmMM1 protein. ZmMM1 acts as a transcription repressor and negatively regulates the transcription of specific target genes, including ZmMM1-target gene 3 (ZmMT3), which functions as a negative regulator of plant immunity and associated cell death. The successful isolation of the ZmMM1 resistance gene will help not only in developing broad-spectrum and durable disease resistance but also in understanding the molecular mechanisms underlying MDR.

A dominant spotted leaf gene TaSpl1 activates endocytosis and defense-related genes causing cell death in the absence of dominant inhibitors

The spotted leaf lesion mimic trait simulates cell death in a plant responding to pathogen infection. Some spotted leaf genes are recessive, while others are dominant. A small number of plants with a lesion mimic phenotype appeared in a segregating population obtained by crossing two normal green wheat strains, XN509 and N07216. Here, we clarified the genetic model and its breeding value. Phenotyping of the consecutive progeny populations over six cropping seasons showed that the spotted leaf lesion mimic phenotype was controlled by a dominant gene designated TaSpl1, which was inhibited by two other dominant genes, designated TaSpl1-I1 and TaSpl1-I2. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed KASP-PCR markers, the TaSpl1 and TaSpl1-I1 loci in N07216 were mapped to the end of chromosomes 3DS and 3BS, respectively. Plants with the spotted phenotype showed lower levels of stripe rust and powdery mildew than those with the normal green phenotype. Compared with normal leaves, the differentially expressed genes in spotted leaves were significantly enriched in plant-pathogen interaction and endocytosis pathways. There were no differences in the yield parameters of the F5 and F6 sister lines, N13039S with TaSpl1 and N13039 N without TaSpl1. These results provide a greater understanding of spotted leaf phenotyping and the breeding value of the lesion mimic allele in developing disease-resistance varieties.

Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice

The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

Gene Mapping, Genome-Wide Transcriptome Analysis, and WGCNA Reveals the Molecular Mechanism for Triggering Programmed Cell Death in Rice Mutant pir1.

Programmed cell death (PCD) is involved in plant growth and development and in resistance to biotic and abiotic stress. To understand the molecular mechanism that triggers PCD, phenotypic and physiological analysis was conducted using the first three leaves of mutant rice PCD-induced-resistance 1(pir1) and its wild-type ZJ22. The 2nd and 3rd leaves of pir1 had a lesion mimic phenotype, which was shown to be an expression of PCD induced by H2O2-accumulation. The PIR1 gene was mapped in a 498 kb-interval between the molecular markers RM3321 and RM3616 on chromosome 5, and further analysis suggested that the PCD phenotype of pir1 is controlled by a novel gene for rice PCD. By comparing the mutant with wild type rice, 1679, 6019, and 4500 differentially expressed genes (DEGs) were identified in the three leaf positions, respectively. KEGG analysis revealed that DEGs were most highly enriched in phenylpropanoid biosynthesis, alpha-linolenic acid metabolism, and brassinosteroid biosynthesis. In addition, conjoint analysis of transcriptome data by weighted gene co-expression network analysis (WGCNA) showed that the turquoise module of the 18 identified modules may be related to PCD. There are close interactions or indirect cross-regulations between the differential genes that are significantly enriched in the phenylpropanoid biosynthesis pathway and the hormone biosynthesis pathway in this module, which indicates that these genes may respond to and trigger PCD.

Hydroperoxide lyase modulates defense response and confers lesion-mimic leaf phenotype in soybean (Glycine max (L.) Merr.).

Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are two important members of P450 enzymes metabolizing hydroperoxy fatty acid to produce jasmonates and aldehydes respectively, which function in response to diverse environmental and developmental stimuli. However, their exact roles in soybean have not been clarified. In present study, we identified a lesion-mimic mutant in soybean named NT302, which exhibits etiolated phenotype together with chlorotic and spontaneous lesions on leaves at R3 podding stage. The underlying gene was identified as GmHPL encoding hydroperoxide lyase by map-based cloning strategy. Sequence analysis demonstrated that a single nucleotide mutation created a premature termination codon (Gln20-Ter), which resulted in a truncated GmHPL protein in NT302. GmHPL RNA was significantly reduced in NT302 mutant, while genes in AOS branch of the 13-LOX pathway were up-regulated in NT302. The mutant exhibited higher susceptibility to bacterial leaf pustule (BLP) disease, but increased resistance against common cutworm (CCW) pest. GmHPL was significantly induced in response to MeJA, wounding, and CCW in wild type soybean. Virus induced gene silencing (VIGS) of GhHPL genes gave rise to similar lesion-mimic leaf phenotypes in upland cotton, coupled with upregulation of the expression of JA biosynthesis and JA-induced genes. Our study provides evidence that competition exist between HPL and AOS branches in 13-LOX pathway of the oxylipin metabolism in soybean, thereby plays essential roles in modulation of plant development and defense.

A novel glycine-rich domain protein, GRDP1, functions as a critical feedback regulator for controlling cell death and disease resistance in rice.

Lesion mimic mutants constitute a valuable genetic resource for unraveling the signaling pathways and molecular mechanisms governing the programmed cell death and defense responses of plants. Here, we identified a lesion mimic mutant, spl-D, from T-DNA insertion rice lines. The mutant exhibited higher accumulation of H2O2, spontaneous cell death, decreased chlorophyll content, up-regulation of defense-related genes, and enhanced disease resistance. The causative gene, OsGRDP1, encodes a cytosol- and membrane-associated glycine-rich domain protein. OsGRDP1 was expressed constitutively in all of the organs of the wild-type plant, but was up-regulated throughout plant development in the spl-D mutant. Both the overexpression and knockdown (RNAi) of OsGRDP1 resulted in the lesion mimic phenotype. Moreover, the intact-protein level of OsGRDP1 was reduced in the spotted leaves from both overexpression and RNAi plants, suggesting that the disruption of intact OsGRDP1 is responsible for lesion formation. OsGRDP1 interacted with an aspartic proteinase, OsAP25. In the spl-D and overexpression plants, proteinase activity was elevated, and lesion formation was partially suppressed by an aspartic proteinase inhibitor. Taken together, our results reveal that OsGRDP1 is a critical feedback regulator, thus contributing to the elucidation of the mechanism underlying cell death and disease resistance.

Melatonin metabolism, signaling and possible roles in plants.

Melatonin is a multifunctional biomolecule found in both animals and plants. In this review, the biosynthesis, levels, signaling, and possible roles of melatonin and its metabolites in plants is summarized. Tryptamine 5-hydroxylase (T5H), which catalyzes the conversion of tryptamine into serotonin, has been proposed as a target to create a melatonin knockout mutant presenting a lesion-mimic phenotype in rice. With a reduced anabolic capacity for melatonin biosynthesis and an increased catabolic capacity for melatonin metabolism, all plants generally maintain low melatonin levels. Some plants, including Arabidopsis and Nicotiana tabacum (tobacco), do not possess tryptophan decarboxylase (TDC), the first committed step enzyme required for melatonin biosynthesis. Major melatonin metabolites include cyclic 3-hydroxymelatonin (3-OHM) and 2-hydroxymelatonin (2-OHM). Other melatonin metabolites such as N1 -acetyl-N2 -formyl-5-methoxykynuramine (AFMK), N-acetyl-5-methoxykynuramine (AMK) and 5-methoxytryptamine (5-MT) are also produced when melatonin is applied to Oryza sativa (rice). The signaling pathways of melatonin and its metabolites act via the mitogen-activated protein kinase (MAPK) cascade, possibly with Cand2 acting as a melatonin receptor, although the integrity of Cand2 remains controversial. Melatonin mediates many important functions in growth stimulation and stress tolerance through its potent antioxidant activity and function in activating the MAPK cascade. The concentration distribution of melatonin metabolites appears to be species specific because corresponding enzymes such as M2H, M3H, catalases, indoleamine 2,3-dioxygenase (IDO) and N-acetylserotonin deacetylase (ASDAC) are differentially expressed among plant species and even among different tissues within species. Differential levels of melatonin and its metabolites can lead to differential physiological effects among plants when melatonin is either applied exogenously or overproduced through ectopic overexpression.