Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.     

<Emphasis Type="Italic">OsBIMK1</Emphasis>, a rice MAP kinase gene involved in disease resistance responses

The activation of mitogen-activated protein kinases (MAPKs) has been previously implicated in signal transduction during plant responses to pathogen attack as well as to various environmental stresses. We have isolated from rice a new MAPK cDNA, OsBIMK1 ( O ryza s ativa L. BTH-induced MAPK 1), which encodes a 369-amino-acid protein with moderate to high nucleotide sequence similarity to previously reported plant MAPK genes. OsBIMK1 contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. We analyzed in detail the expression of OsBIMK1 upon treatment with various chemical and biological inducers of resistance responses in rice and in both incompatible and compatible interactions between rice and Magnaporthe grisea. Expression of OsBIMK1 was activated rapidly upon treatment with benzothiadiazole (BTH) as well as with dichloroisonicotinic acid, probenazole, jasmonic acid and its methyl ester, Pseudomonas syringae pv. syringae, or wounding. Expression of OsBIMK1 was induced rapidly during the first 36 h after inoculation with M. grisea in BTH-treated rice seedlings and in an incompatible interaction between M. grisea and a blast-resistant rice genotype. BTH treatment induced a systemic activation of OsBIMK1 expression. These results suggest that OsBIMK1 plays an important role in rice disease resistance.     

Cold-active winter rye glucanases with ice-binding capacity

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic beta-1,3-glucanases and an acidic beta-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%-35% at -4 degrees C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye beta-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one beta-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

差异显示法分离水稻抗稻瘟病相关基因

采用mRNA差异显示技术,分析水稻稻瘟病抗源材料“地谷”叶片受稻瘟病菌侵染前后的基因的表达差异,获得87个差异片段。对这87个差异片段进行了回收、重扩增与克隆,并对其中的81个片段进行了杂交鉴定。斑点杂交结果证实其中6个片段受稻瘟病菌诱导表达。进一步克隆测序并进行数据库比对分析表明其中一个与水稻4号染色体中一推测的苹果酸合成酶高度同源,一个与水稻11号染色体上的RPR1基因高度同源,RPR1基因具有保守的NBS-LRR结构,并与水稻防卫反应的信号传导有关;另一个与水稻第6号染色体上一推测的硫氧还蛋白高度同源,其余3个为新的cDNA片段。     

Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of omega-3 fatty acid desaturases

Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZ1 and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.     

Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. In Arabidopsis, 20 MAPKs have been identified and divided into four major groups. In rice, a monocot model and economically important cereal crop, only five MAPKs were characterized, including three related to the host defense response. In this study, we have identified 17 members of the rice MAPK gene (OsMPK) family through an in silico search of rice genome databases. Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, we propose that MAPKs can be divided into six groups. Interestingly, the rice genome contains many more MAPKs with the TDY phosphorylation site (11 members) than with the TEY motif (six members). In contrast, the Arabidopsis genome contains more MAPKs with the TEY motif (12 members) than with the TDY motif (eight members). Upon inoculation with the blast fungus (Magnaporthe grisea), nine of 17 OsMPK genes were found to be induced at the mRNA level during either early, late, or both stages of infection. Four of the M. grisea-induced OsMPK genes were associated with host-cell death in the lesion-mimic rice mutant, and eight of them were differentially induced in response to defense signal molecules such as jasmonic acid, salicylic acid, abscisic acid, and ethylene. The genome-wide expression analysis suggests that about half of the rice MAPK genes are associated with pathogen infection and host defense response.     

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.     

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection.     

The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress.

14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins.     

A rice ��-1,3-glucanase gene Osg1 is required for callose degradation in pollen development

Plant β-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative β-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these β-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.     

Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Functional divergence in the Arabidopsis beta-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states

Plant beta-1,3-glucanases (beta-1,3-Gs) (E.C. 3.2.1.39) comprise large, highly complex gene families involved in pathogen defense as well as a wide range of normal developmental processes. In spite of previous phylogenetic analyses that classify beta-1,3-Gs by sequence relatedness, the functional evolution of beta-1,3-Gs remains unclear. Here, expression and phylogenetic analyses have been integrated in order to investigate patterns of functional divergence in the Arabidopsis beta-1,3-G gene family. Fifty beta-1,3-G genes were grouped into expression classes through clustering of microarray data, and functions were inferred based on knowledge of coexpressed genes and existing literature. The resulting expression classes were mapped as discrete states onto a phylogenetic tree and parsimony reconstruction of ancestral expression states was performed, providing a model of expression divergence. Results showed a highly nonrandom distribution of developmental expression states in the phylogeny (P = 0.0002) indicating a significant degree of coupling between sequence and developmental expression divergence. A weaker, yet significant level of coupling was found using stress response data, but not using hormone-response or pathogen-response data. According to the model of developmental expression divergence, the ancestral function was most likely involved in cell division and/or cell wall remodeling. The associated expression state is widely distributed in the phylogeny, is retained by over 25% of gene family members, and is consistent with the known functions of beta-1,3-Gs in distantly related species and gene families. Consistent with previous hypotheses, pathogenesis-related (PR) beta-1,3-Gs appear to have evolved from ancestral developmentally regulated beta-1,3-Gs, acquiring PR function through a number of evolutionary events: divergence from the ancestral expression state, acquisition of pathogen/stress-responsive expression patterns, and loss of the C-terminal region including the glycosylphosphatidylinisotol (GPI)-anchoring site thus allowing for extracellular secretion.