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.     

OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea

Calcium-dependent protein kinases are important decoders of calcium signals in plants, which are involved in plant immunity. We report isolation and functional characterization of a pathogen-responsive OsCPK20 gene in rice. The expression of OsCPK20 in rice was significantly induced following treatment with a Magnaporthe grisea elicitor. Overexpression of constitutively active OsCPK20 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK20 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic Arabidopsis and rice was associated with activated expression of both SA- and JA-related defense genes. We also found that OsCPK20 was significantly induced by drought stress, indicating that OsCPK20 might be involved in plant response to drought stress. Taken together, our results indicate that rice OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against M. grisea, and that it may enhance disease resistance by activating both SA- and JA-dependent defense responses.     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999     

Expression of an elicitor-encoding gene from <Emphasis Type="Italic">Magnaporthe grisea</Emphasis> enhances resistance against blast disease in transgenic rice

Elicitors are molecules that stimulate defense responses in plants. Previously, an elicitor-encoding gene, named pemG1, was isolated from Magnaporthe grisea. To assess the function of pemG1 in rice (Oryza sativa L. cv. Nipponbare), the gene was cloned under a constitutive maize ubiquitin promoter and introduced into Nipponbare cultivar. The resultant plants showed stable integration and constitutive expression of the pemG1 gene. The expression of defense-related gene for phenylalanine ammonia-lyase was triggered and proline content was also increased in pemG1-expressing plants. The pemG1-expressing plants showed enhanced resistance against rice blast after inoculation with M. grisea spores, suggesting that the pemG1 expression enhances disease resistance in transgenic rice. DQ and JM contributed equally to this paper.     

Expression of a plant defensin in rice confers resistance to fungal phytopathogens

Transgenic rice (Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs-AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. Expression levels of Rs-AFP2 ranged from 0.45 to 0.53% of total soluble protein in transgenic plants. It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. No effect on plant morphology was observed in the Rs-AFP2 expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of Rs-AFP2 plants on the in vitro growth of M. oryzae indicated that the Rs-AFP2 protein produced by transgenic rice plants was biologically active. Transgene expression of Rs-AFP2 was not accompanied by an induction of pathogenesis-related (PR) gene expression, suggesting that the expression of Rs-AFP2 directly inhibits the pathogens. Here, we demonstrate that transgenic rice plants expressing the Rs-AFP2 gene show enhanced resistance to M. oryzae and R. solani, two of the most important pathogens of rice.     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

Infection-related development in the rice blast fungus Magnaporthe grisea

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.     

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R₀ C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R₀ generation, suggesting gene silencing. Preliminary screening of these R₁ plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid

Linoleic acid (18:2) and linolenic acid (18:3) are sources for various oxidized metabolites called oxylipins, some of which inhibit growth of fungal pathogens. In a previous study, we found disease resistance to rice blast fungus Magnaporthe grisea enhanced in 18:2-accumulating transgenic rice (F78Ri) in which the conversion from 18:2 to 18:3 was suppressed. Here, we demonstrate that 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibit growth of M. grisea more strongly than their 18:3-derived counterparts. Furthermore, in F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results suggest that the increased accumulation of antifungal oxylipins, such as HPODEs and HODEs, causes the enhancement of disease resistance against M. grisea.     

Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four antifungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R₁ and R₂ progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F₁ plants, resulting from a cross of R₂ homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).     

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

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.