A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity

To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.     

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9 kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four α-helixes and 16 β-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2 h after initiation of the stress and was highly induced between 18–72 h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.     

Stress-induced expression and structure of the putative gene hyp-1 for hypericin biosynthesis

The phenolic oxidative coupling protein (Hyp-1) with proposed activity in the biosynthesis of hypericin in Hypericum perforatum shares about 50 % sequence similarity with Bet.v.1-like/PR-10 proteins. In our previous study, we showed that this protein is not a limiting factor in hypericin biosynthesis. To ascertain the role of Hyp-1 in defense mechanisms, we have analyzed some structural features of the hyp-1 gene in 14 Hypericum species with different abilities to synthesise hypericin. We show that the hyp-1 gene possesses characteristics typical for genes encoding plant PR-10 proteins. The coding sequence of the hyp-1 gene is interrupted by a single 86- to 125-bp intron localised strictly in codon 62, which is a typical feature of the dicot PR-10 subfamily. The localisation of the intron is conserved in all 14 tested Hypericum species indicating a common evolutionary history with genes encoding PR-10 proteins. In addition, we report that the hyp-1 gene exhibits a similar response to stress conditions as the PR-10 proteins encoding genes. Following either wounding or infection by Agrobacterium tumefaciens, all analysed Hypericum species exhibited rapid and significant upregulation of hyp-1 gene expression; this was particularly observed in hypericin-producing species. On the other hand, in the presence of high levels of abscisic acid, different levels of gene expression were observed.     

Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco

A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses.     

Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis-related (PR) proteins such as the acidic PR-1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco - elevated H₂O₂ levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA-effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR-1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA-mediated induction of PR-1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K-252a and staurosporine induced PR-1 gene expression. PR-1 gene induction by K-252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K-252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR-1 gene induction. The SA-inducible acidic PR-2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.     

The Fungal Pathogen Moniliophthora perniciosa Has Genes Similar to Plant PR-1 That Are Highly Expressed during Its Interaction with Cacao

The widespread SCP/TAPS superfamily (SCP/Tpx-1/Ag5/PR-1/Sc7) has multiple biological functions, including roles in the immune response of plants and animals, development of male reproductive tract in mammals, venom activity in insects and reptiles and host invasion by parasitic worms. Plant Pathogenesis Related 1 (PR-1) proteins belong to this superfamily and have been characterized as markers of induced defense against pathogens. This work presents the characterization of eleven genes homologous to plant PR-1 genes, designated as MpPR-1, which were identified in the genome of Moniliophthora perniciosa, a basidiomycete fungus responsible for causing the devastating witches'' broom disease in cacao. We describe gene structure, protein alignment and modeling analyses of the MpPR-1 family. Additionally, the expression profiles of MpPR-1 genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the MpPR-1 family in the conditions analyzed. Interestingly, some of them were highly and specifically expressed during the interaction of the fungus with cacao, suggesting a role for the MpPR-1 proteins in the infective process of this pathogen. Hypothetical functions assigned to members of the MpPR-1 family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of PR-1-like genes for fungal virulence on plants.     

Trigalactosyldiacylglycerol 3 protein orthologs are required for basal disease resistance in Nicotiana benthamiana

Phosphatidic acid plays an important role in Nicotiana benthamiana immune responses against phytopathogenic bacteria. We analyzed the contributions of endoplasmic reticulum-derived chloroplast phospholipids, including phosphatidic acid, to the resistance of N. benthamiana against Ralstonia solanacearum. Here, we focused on trigalactosyldiacylglycerol 3 (TGD3) protein as a candidate required for phosphatidic acid signaling. On the basis of Arabidopsis thaliana TGD3 sequences, we identified two putative TGD3 orthologs in the N. benthamiana genome, NbTGD3-1 and NbTGD3-2. To address the role of TGD3s in plant defense responses, we created double NbTGD3-silenced plants using virus-induced gene silencing. The NbTGD3-silenced plants showed a moderately reduced growth phenotype. Bacterial growth and the appearance of bacterial wilt disease were accelerated in NbTGD3-silenced plants, compared with control plants, challenged with R. solanacearum. The NbTGD3-silenced plants showed reduced both expression of allene oxide synthase that encoded jasmonic acid biosynthetic enzyme and NbPR-4, a marker gene for jasmonic acid signaling, after inoculation with R. solanacearum. Thus, NbTGD3-mediated endoplasmic reticulum—chloroplast lipid transport might be required for jasmonic acid signaling-mediated basal disease resistance in N. benthamiana.     

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.     

Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

Early signal transduction linking the synthesis of jasmonic acid in plant

Jasmonate signaling plays a critical role in protecting plants from pathogens or insect attacks and in limiting damage from abiotic stress. Many events contribute to the regulation of jasmonic acid (JA) synthesis during abiotic or biotic stress, but the details of the underlying mechanism remain unclear. In this Mini-Review paper, we discuss the possible roles of reactive oxygen species (ROS), nitric oxide (NO), calcium influx and mitogen-activated protein kinase (MAPK) cascade during JA synthesis or JA signal transduction.Key words: jasmonic acid, singal, transductionJasmonic acid (JA) is a member of the jasmonate group of plant hormones; it is biosynthesized from linolenic acid by the octadecanoid pathway.¹ The main functions of this hormone are growth related, including growth inhibition, senescence and leaf abscission. It also plays an important role in plant response to wounding and in systemic resistance. JA has a structure similar to that of mammal prostaglandins and is synthesized from alpha-linolenic acid, which is a C-18 poly-unsaturated fatty acid. Lipoxygenase, allene oxide synthase and allene oxide cyclase are the putative key enzymes for JA synthesis; these enzymes have chloroplast transit peptides that direct their import into chloroplasts. JA can be conjugated with amino acids, namely, leucine, valine, isoleucine and the sugar, B-glucoside using UDP-glucose. (-)-JA and (-)-methyl jasmonate are major JAs in plants. Methyl jasmonate (MeJA) in particular is a strong candidate for airborne signals that mediate interplant communication for defense responses. JA and its derivates induce the production of vegetative storage proteins, osmotin, thionin (antifungal) and defensin. It also induces enzymes related to phytoalexin, chalcone synthase, phenylalanine ammonia lyase (PAL), and hydroxymethylglutaryl-COA reductase; it also induces protease inhibitors to suppress the insect growth. JA and ethylene induce PR-3, PR-4 and PDF 1.2 chitinases (CHI-B) and hevein-like protein. In plants, ROS, Calcium ion influx, MAP kinase cascade, and NO, a novel signaling molecule are involved in the JA octadecanoid signal pathway.^1–4     

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial‐associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti‐microbial secondary metabolites. Mitogen‐activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor‐induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4^DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4^DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4^DD‐induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP‐triggered defense responses.     

Microarray Analysis of Tomato’s Early and Late Wound Response Reveals New Regulatory Targets for Leucine Aminopeptidase A

Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A’s role in modulation of wound responses extends beyond the well described late-wound gene core.     

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

Background

Plants have two related immune systems to defend themselves against pathogen attack. Initially, pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses.

Results

We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling.

Conclusions

Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.     

A nonspecific lipid transfer protein,StLTP10, mediates resistance to Phytophthora infestans in potato

Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.