Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens.

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae.

The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.

Meroterpenes from Psoralea corylifolia against Pyricularia oryzae.

Six new meroterpenes, namely, 13-methoxyisobakuchiol (1), 13-ethoxyisobakuchiol (2), 12,13-dihydro-13-hydroxybakuchiol (3), Δ(10)-12,13-dihydro-12-(R,S)-methoxyisobakuchiol (4 and 5), and 15-demetyl-12,13-dihydro-13-ketobakuchiol (6), together with four known ones, namely, Δ(3),2-hydroxybakuchiol (7), Δ(1),3-hydroxybakuchiol (8), bakuchiol (9), and Δ(1,3)-bakuchiol (10), were isolated from the seeds of Psoralea corylifolia. Their structures were identified based on spectral data, including those obtained via 1D and 2D NMR, and MS spectral analyses. Antifungal screening results indicated that all compounds showed moderate inhibitory activities against Pyricularia oryzae.

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

A novel protein Com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.

In Magnaporthe oryzae, pyriform conidia are the primary inoculum and the main source for disease dissemination in the field. In this study, we identified and characterized the COM1 gene that was disrupted in three insertional mutants producing slender conidia. COM1 encodes a putative transcription regulator unique to filamentous ascomycetes. The com1 disruption and deletion mutants had similar defects in conidium morphology and were significantly reduced in virulence on rice and barley seedlings. Microscopic examination revealed that the Deltacom1 mutants were defective in appressorium turgor generation, penetration, and infectious growth. COM1 was expressed constitutively in M. oryzae. The Com1 protein had putative helix-loop-helix structures and three predicted nuclear localization signal sequences. In transformants expressing COM1(335-613)-enhanced green fluorescent protein fusion constructs, fluorescence signals were observed in the nucleus. Our data indicated that the COM1 gene may encode a novel transcription regulator that regulates conidial development and invasive growth in M. oryzae.

Molecular cloning and characterization of a rice blast-inducible RING-H2 type zinc finger gene.

A novel blast-inducible RING-H2 type zinc finger protein gene OsRING-1 was cloned from rice by cDNA library screening. OsRING-1 is 1670 bp in length and encodes a 46.6 kDa basic protein with two transmembrane (TM) domains, a basic domain (BD), a conserved domain (CD), a RING finger domain and a serine rich (S-rich) domain. By database search, OsRING-1 was mapped on chromosome 2 and clustered together with other six zinc finger genes. The promoter sequence analysis of OsRING-1 gene revealed that some ABA, GA, ethylene, wound, drought, heat stress and pathogen infection responsive elements were found within the OsRING-1 promoter region. Northern analysis showed that OsRING-1 was induced in different degree by pathogen infections, SA, ABA, JA and ethephon (ET) treatments. Tissue expression analysis showed that OsRING-1 was constitutively strongly expressed in roots, but faintly in stems, leaves and sheaths. Taken together, OsRING-1, as a novel C3H2C3-type zinc finger protein involved in many stress responses in rice might plays a role as a transcription regulator in plant stress response signal transduction pathways.

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.

Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress.

The Woronin body is a peroxisome-derived dense-core vesicle that is specific to several genera of filamentous ascomycetes, where it has been shown to seal septal pores in response to cellular damage. The Hexagonal peroxisome (Hex1) protein was recently identified as a major constituent of the Woronin body and shown to be responsible for self-assembly of the dense core of this organelle. Using a mutation in the Magnaporthe grisea HEX1 ortholog, we define a dual and essential function for Woronin bodies during the pathogenic phase of the rice blast fungus. We show that the Woronin body is initially required for proper development and function of appressoria (infection structures) and subsequently necessary for survival of infectious fungal hyphae during invasive growth and host colonization. Fungal mycelia lacking HEX1 function were unable to survive nitrogen starvation in vitro, suggesting that in planta growth defects are a consequence of the mutant's inability to cope with nutritional stress. Thus, Woronin body function provides the blast fungus with an important defense against the antagonistic and nutrient-limiting environment encountered within the host plant.

Cerebroside elicitors found in diverse phytopathogens activate defense responses in rice plants.

Cerebrosides A and C, compounds categorized as glycosphingolipids, were isolated in our previous study from the rice blast fungus (Magnaporthe grisea) as novel elicitors which induce the synthesis of rice phytoalexins. In this paper, these cerebroside elicitors showed phytoalexin-inducing activity when applied to plants by spray treatment and also induced the expression of pathogenesis-related (PR) proteins in rice leaves. This elicitor activity of the cerebrosides showed the structural specificity as that for the induction of phytoalexins. Ceramides prepared from the cerebrosides by removal of glucose also showed the elicitor activity even in lower level compared to the cerebrosides. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus, an economically devastating agent of disease of rice in Japan. The cerebrosides elicitors protected rice plants from other disease as well and were found to occur in a wide range of different phytopathogens, indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions.

Cyclopenta[b]benzofurans from Aglaia species with pronounced antifungal activity against rice blast fungus (Pyricularia grisea).

Eight flavaglines, six cyclopenta[b]benzofurans, a cyclopenta[bc]benzopyran, and a benzo[b]oxepine, together with an aromatic butyrolactone were isolated from Aglaia odorata, A. elaeagnoidea, and A. edulis (Meliaceae) and tested against the three plant pathogens Pyricularia grisea, Fusarium avenaceum, and Alternaria citri for antifungal properties. Using the microdilution technique linked with digital image analysis of germ tubes, the benzofurans displayed strong activity, whereas the benzopyran, benzoxepine, and butyrolactone were inactive at the highest concentration tested. P. grisea, responsible for rice blast disease, was the most susceptible fungus against all benzofurans, with rocaglaol as the most active derivative. Based on EC(50), EC(90), and MIC values, the antifungal activity of rocaglaol was clearly higher than of the reference compounds, blasticidin S and Benlate.

Lesion mimic mutants of rice with alterations in early signaling events of defense.

We screened 93 lesion mimic mutants of rice for resistance to the blast fungus, Magnaporthe grisea, and found eight mutants that exhibited significant resistance to the fungus. We called these mutants cdr (cell death and resistance) and further analyzed three of them. Two mutations, cdr1 and cdr2, were recessive and one, Cdr3, was dominant. Many small brownish lesions developed over the entire leaf of the mutants 20-50 days after sowing. TUNEL staining revealed that DNA fragmentation occurred in leaf blade cells of the homozygous Cdr3 mutants. Autofluorescence and callose deposition were visible in leaf cells of these three mutants. Activation of two defense-related genes, PBZ1 and PR1, was observed in the leaves of the mutants; high expression of PBZ1 was correlated with the lesion formation in the three mutants, whereas PR1 was constitutively expressed in the cdr2 and Cdr3 mutants irrespective of the lesion formation. Levels of momilactone A, a major phytoalexin of rice, in these mutants were increased approximately 100-400-fold relative to the wild-type levels. Suspension-cultured cells of the cdr1 and cdr2 but not Cdr3 produced higher levels of H2O2 than the wild type when treated with calyculin A, an inhibitor of protein phosphatase 1. These results suggest that biochemical lesions of cdr1 and cdr2 lie in the early signaling steps leading to activation of the NADPH oxidase and that type-1 protein phosphatase is operative in protein dephosphorylation involved in NADPH oxidase activation.