Effects of Colonization of a Bacterial Endophyte,Azospirillum sp. B510, on Disease Resistance in Rice

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.     

The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H₂O₂ production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.     

Rice <Emphasis Type="Italic">SERK1</Emphasis> gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection

Here we report on the isolation and characterization of a somatic embryogenesis receptor-like kinase (OsSERK1) gene in rice (Oryza sativa). The OsSERK1 gene belongs to a small subfamily of receptor-like kinase genes in rice and shares a highly conserved gene structure and extensive sequence homology with previously reported plant SERK genes. Though it has a basal level of expression in various rice organs/tissues, as high expression level was detected in rice callus during somatic embryogenesis. Suppression of OsSERK1 expression in transgenic calli by RNA interference resulted in a significant reduction of shoot regeneration rate (from 72% to 14% in the japonica rice Zhonghua11). Overexpression of OsSERK1, however, increased the shoot regeneration rate (from 72% to 86%). Interestingly, OsSERK1 is significantly activated by the rice blast fungus, particularly during the incompatible interaction, and is associated with host cell death in Sekigushi lesion mimic mutants. This gene is also inducible by defense signaling molecules such as salicylic acid, jasmonic acid, and abscisic acid. Furthermore, constitutive overexpression of OsSERK1 in two rice cultivars led to an increase in host resistance to the blast fungus. Our data suggest that OsSERK1 may partially mediate defense signal transduction in addition to its basic role in somatic embryogenesis.     

OsRPK1, a novel leucine-rich repeat receptor-like kinase,negatively regulates polar auxin transport and root development in rice

Background

Leucine-rich-repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of putative RLKs in plants. Although several members in this subfamily have been identified, the studies about the relationships between LRR-RLKs and root development are still few. We previously identified a novel LRR-RLK in rice roots, and named it OsRPK1.

Methods

In this study, we first detected OsRPK1 kinase activity in vitro, and assessed its expression profile. We then investigated its biological function using transgenic rice plants over- and under-expressing OsRPK1.

Results

The OsRPK1 gene, which encodes a Ca^2 +-independent Ser/Thr kinase, was predominantly expressed in root tips, leaf blades, and undifferentiated suspension cells, and was markedly induced by treatment with auxin or ABA. Knockdown of OsRPK1 promoted the growth of transgenic rice plants, and increased plant height and tiller numbers. In contrast, over-expressing plants showed undeveloped adventitious roots, lateral roots, and a reduced root apical meristem. OsRPK1 over-expression also inhibited the expression of most auxin efflux carrier OsPIN genes, which was accompanied by changes in PAT and endogenous free IAA distribution in the leaves and roots.

Conclusions

The data indicated that OsRPK1, a novel leucine-rich-repeat receptor-like kinase, affects the root system architecture by negatively regulating polar auxin transport in rice.

General significance

This study demonstrated a common regulatory pathway of root system development in higher plants, which might be initiated by external stimuli via upstream receptor-like kinases and downstream carriers for polar auxin transport.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.     

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.     

Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Transgenic indica rice expressing Mirabilis jalapa antimicrobial protein (Mj-AMP2) shows enhanced resistance to the rice blast fungus Magnaporthe oryzae

Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Identification and characterization of 2'-deoxyuridine from the supernatant of conidial suspensions of rice blast fungus as an infection-promoting factor in rice plants

We previously detected infection-promoting activity in the supernatant of the conidial suspension (SCS) of the rice blast fungus. In the present study, a molecule carrying the activity was purified and identified as 2'-deoxyuridine (dU). The infection-promoting activity of dU was strictly dependent on its chemical structure and displayed characteristics consistent with those of the SCS. Notably, the activity of dU was exclusively detected during interactions between rice and virulent isolates of the fungus, the number of susceptible lesions in leaf blades was increased by dU, and nonhost resistance in rice plants was not affected by treatment with dU. In addition, the expression of pathogensis-related genes, accumulation of H(2)O(2), and production of phytoalexins in rice in response to inoculation with virulent fungal isolates was not suppressed by dU. The infection-promoting activity of dU was not accompanied by elevated levels of endogenous abscissic acid, which is known to modify plant-pathogen interactions, and was not detected in interactions between oat plants and a virulent oat blast fungus isolate. Taken together, these results demonstrate that dU is a novel infection-promoting factor that acts specifically during compatible interactions between rice plants and rice blast fungus in a mode distinct from that of toxins and suppressors.     

RMo1 confers blast resistance in barley and is located within the complex of resistance genes containing Mla, a powdery mildew resistance gene

Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54-20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53-33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53-33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.     

Genetic Analysis of Resistance to Rice Blast: A Study on the Inheritance of Resistance to the Blast Disease Pathogen in an F3 Population of Rice

Blast caused by the fungus Magnaporthae grisea (Herbert) Borr. (anamorphe Pyricularia oryza Cav.) is a serious disease of rice (Oryza sativa L.). One method to overcome this disease is to develop disease resistant cultivars. Due to the genetic plasticity in the pathogen genome, there is a continuous threat to the effectiveness of the developed cultivars. Additional studies of the genetics of resistance, virulence stability and functional genomics are required to accelerate research into understanding the molecular basis of blast disease resistance. In this study, individual plants of the F₃ population derived from Pongsu Seribu 2 and Mahsuri were used for pathogenesis assays and inheritance studies of blast resistance. The study was performed with two of the most virulent Malaysian M. grisea pathotypes: P7.2 and P5.0. For blast screening, plants were scored based on the IRRI Standard Evaluation System (SES). F₃ populations showed a segregation ratio of 3R:1S for pathotype P7.2, indicating that resistance to this pathotype is likely controlled by a single nuclear gene. Chi‐square analysis showed that the F₃ families segregated in a 15R:1S ratio for pathotype P5.0. Therefore, locus interactions or epitasis of blast resistance occur against pathotype P5.0 in the F₃ population derived from Pongsu Seribu 2 and Mahsuri. This can be explained by the presence of two independent dominant genes that when present simultaneously, provide resistance to the M. gresia pathotype P5.0. These results indicated that blast resistance in rice is due to the combined effects of multiple loci with major and minor effects. The genetic data generated here will be useful in the breeding of local cultivars for resistance to field blast. The methodology reported here will facilitate the mapping of genes and quantitative trait loci (QTLs) underlying the blast resistance trait.     

Overexpression of rice <Emphasis Type="Italic">LRK1</Emphasis> restricts internode elongation by down-regulating <Emphasis Type="Italic">OsKO2</Emphasis>

Rice (Oryza sativa) has the potential to undergo rapid internodal elongation which determines plant height. Gibberellin is involved in internode elongation. Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants. LRR-RLKs play important functions in mediating a variety of cellular processes and regulating responses to environmental signals. LRK1, a PSK receptor homolog, is a member of the LRR-RLK family. In the present study, differences in ectopic expression of LRK1 were consistent with extent of rice internode elongation. Analyses of gene expression demonstrated that LRK1 restricts gibberellin biosynthesis during the internode elongation process by down-regulation of the gibberellin biosynthetic gene coding for ent-kaurene oxidase.     

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.     

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom,Confers Resistance to the Blast Fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.