<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.     

Molecular cloning and expression analysis of rice OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins.

We describe the cloning and identification of a rice cDNA, OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins. OsTVLP1 has an open reading frame of 2955 bp, which encodes a 984 amino acid protein, containing a citron homology (CNH) domain at its N-terminal and a clathrin heavy-chain repeat homology (CLH) domain at its C-terminal. The expression of OsTVLP1 was induced by treatments with benzothiadiazole (BTH), a chemical activator of plant disease resistance responses, and by infection of the blast fungus, Magnaporthe grisea. Importantly, the expression of OsTVLP1 was activated specifically in disease resistance response induced by BTH and in an incompatible interaction between rice and the blast fungus. Our observations suggest that OsTVLP1 may play a role in rice disease resistance response against pathogen infection.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.     

Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences

The rice blast resistance gene Pib is a member of the nucleotide binding site (NBS) and leucine-rich repeat (LRR) class of plant disease resistance (R) genes and belongs to a small gene family. We describe here the isolation and characterization of a Pib homologue (PibH8), and extensive investigation of the expression of the Pib gene family (Pib, PibH8, HPibH8-1, HPibH8-2) under various environmental and chemical treatments. PibH8 shows 42% identity and 60% similarity to Pib and, like Pib, has a duplication of the kinase 1a, 2, and 3a motifs of the NBS region in the N-terminal half of the protein. Interestingly, genes of the Pib family exhibit a diurnal rhythm of expression. RNA gel blot analysis revealed that their expression was regulated dramatically by environmental signals. such as temperature, light and water availability. Their expression was also induced by chemical treatments, such as jasmonic acid, salicylic acid, ethylene and probenazole. Our findings suggest that expression of the Pib gene family is up-regulated by environmental conditions that would favour pathogen infection. This may reflect the evolution of anticipatory control of R gene expression.     

OsBISAMT1, a gene encoding S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, is differentially expressed in rice defense responses

We isolated and identified a full-length cDNA, OsBISAMT1 [Oryza sativa L. benzothiadiazole (BTH)-induced SAMT 1], which encodes a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) from rice. OsBISAMT1 contains an ORE of 1128 bp, which predicts to encode a 375 aa protein. The OsBISAMT1 protein sequence shows a high level of identity to known plant SAMTs and contains a conserved characteristic methyltransferase domain. OsBISAMT1 is a member of a small gene family in the rice genome. Expression of OsBISAMT1 in rice leaves was induced by treatments with benzothiadiazole and salicylic acid, which are capable of inducing rice disease resistance. OsBISAMT1 was also up-regulated in both incompatible and compatible interactions between rice and the blast fungus, Magnaporthe grsiea, but the induced expression of OsBISAMT1 was greater and more rapid in the incompatible interaction than that in the compatible one. Moreover, mechanical wounding also activated OsBISAMT1 expression. The results suggest that OsBISAMT1 may be involved in disease resistance responses as well as in wound response in rice.     

Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.     

Molecular cloning and characterization of a novel SNAP25-type protein gene <Emphasis Type="Italic">OsSNAP32</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The SNAP25-type proteins belong to the superfamily of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and function as important components of the vesical trafficking machinery in eukaryotic cells. In this paper, we report the cloning and expression characterization of OsSNAP32 gene, and the subcellular localization of its encoded protein. The OsSNAP32 gene contains five exons and four introns, and is located between RFLP markers C12276S and S1917 on chromosome 2 in rice. The OsSNAP32 has a molecular weight of 31.3 kD, comprises 283 amino acid residues, and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal, respectively. Multiple sequence alignment of the SNARE domains indicates that OsSNAP32 protein is homologous to HvSNAP34 and HvSNAP28 (63% and 55% of amino acid identity respectively) from barley. The transient expression method in onion epidermal cells, revealed that OsSNAP32 is located in the plasma membrane, like other SNAP25-type proteins. Semi-quantitative RT-PCR assay showed that the OsSNAP32 is highly expressed in leaves and culms, and low in roots of rice, while hardly detected in immature spikes and flowering spikes. The expression of OsSNAP32 was significantly activated in rice seedlings treated with H2O2, PEG6000, and low temperature or after inoculation with rice blast (Magnaporthe grisea strain Hoku 1). The results suggest that this gene belongs to a novel member of this gene family encoding SNAP25-type proteins, involved in the rice responses to biotic and abiotic stresses.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Physical mapping of a rice lesion mimic gene,<Emphasis Type="Italic"> Spl1</Emphasis> , to a 70-kb segment of rice chromosome 12

The rice lesion mimic mutant spotted leaf 1 ( spl1) was first identified in the rice ( Oryza sativa) cultivar Asahi in 1965. This mutant displayed spontaneous disease-like lesions in the absence of any pathogen, and was found to confer resistance to multiple isolates of rice blast. We employed a map-based cloning strategy to localize the Spl1 gene. A total of ten cleaved amplified polymorphic sequence (CAPS) markers linked to the Spl1 gene were identified and mapped to an 8.5-cM region on chromosome 12. A high-resolution genetic map was developed using these ten CAPS markers and a segregating population consisting of 3202 individuals. A BAC contig containing four BAC clones was constructed, and Spl1 was localized to a 423-kb region. Seven spl1 mutants were obtained from the IR64 deletion mutant collection, and molecular analysis using these mutants delimited the Spl1 gene to a 70-kb interval, covered by two BAC clones. These results provide the basis for cloning this gene, which is involved in cell death and disease resistance in rice.Communicated by R. HagemannThe first two authors contributed equally to the work     

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.     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.