Susumu Mochizuki

The Arabidopsis WAVY GROWTH 2 Protein Modulates Root Bending in Response to Environmental Stimuli

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an α/β-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.

Targeted Gene Disruption of OsCERK1 Reveals Its Indispensable Role in Chitin Perception and Involvement in the Peptidoglycan Response and Immunity in Rice

OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.

Stress-induced expression of the transcription factor RERJ1 is tightly regulated in response to jasmonic acid accumulation in rice

The plant hormone jasmonic acid (JA) regulates various developmental processes and plant defence responses to environmental stresses. We previously reported that RERJ1, a JA-inducible transcription factor in rice, is up-regulated by exposure to wounding and drought stress. Here, we demonstrated that the expression of RERJ1 after wounding is regulated in a JA-dependent manner in rice, based on histochemical analysis of RERJ1 promoter-GUS transgenic plants. RERJ1 expression was induced only at the region of injury after wounding, whereas expression was induced in the entire leaf after drought. According to JA measurements of stressed leaves, high accumulation of endogenous JA was only detected around the wound site in a rice leaves, whereas the drought treatment led to uniform accumulation of JA in the entire leaf, suggesting that RERJ1will be a useful marker gene for studies on localization of JA in rice. Nuclear localization and transactivation ability of RERJ1 were also demonstrated. These results suggest that RERJ1 plays a role as a transcriptional activator for regulating stress-inducible gene expression, with a strong correlation to JA accumulation in the stressed region.

The wavy growth 3 E3 ligase family controls the gravitropic response in Arabidopsis roots.

Regulation of the root growth pattern is an important control mechanism during plant growth and propagation. To better understand alterations in root growth direction in response to environmental stimuli, we have characterized an Arabidopsis thaliana mutant, wavy growth 3 (wav3), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The wav3 mutant shows a greater curvature of root bending in response to gravity, but a smaller curvature in response to light, suggesting that it is a root gravitropism-enhancing mutation. This wav3 phenotype also suggests that enhancement of the gravitropic response in roots strengthens root tip impedance after contact with the agar surface and/or causes an increase in subsequent root bending in response to obstacle-touching stimulus in these mutants. WAV3 encodes a protein with a RING finger domain, and is mainly expressed in root tips. RING-containing proteins often function as an E3 ubiquitin ligase, and the WAV3 protein shows such activity in vitro. There are three genes homologous to WAV3 in the Arabidopsis genome [EMBRYO SAC DEVELOPMENT ARREST 40 (EDA40), WAVH1 and WAVH2 ], and wav3 wavh1 wavh2 triple mutants show marked root gravitropism abnormalities. This genetic study indicates that WAV3 functions positively rather than negatively in root gravitropism, and that enhancement of the gravitropic response in wav3 roots is dependent upon the function of WAVH2 in the absence of WAV3. Hence, our results demonstrate that the WAV3 family of proteins are E3 ligases that are required for root gravitropism in Arabidopsis.

Live-cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live‐cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane‐rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High‐resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first‐invaded cell. Furthermore, a newly developed, long‐term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first‐invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection.

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae.

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.

Localization of probe-accessible chitin and characterization of genes encoding chitin-binding domains during rice–Magnaporthe oryzae interactions

We examined the localization of probe-accessible chitin in Magnaporthe oryzae, which causes rice blast disease, during the early infection process and the functions of two genes encoding a chitin-binding domain (ChBD). Invasive hyphae in the first-invaded rice cell showed little staining with fluorescently labeled wheat germ agglutinin, a probe to detect chitin. However, in the second-invaded cell, hyphae showed strong fluorescence, and treatment with chitinase diminished the signal. Fourteen ChBD genes encoding family 18 carbohydrate-binding module (CBM18) were isolated from a Japanese strain of M. oryzae, Ina86-137. Reverse transcription-polymerase chain reaction analysis demonstrated that ChBD-1, ChBD-6, ChBD-8, ChBD-13, and ChBD-15 are expressed in the rice sheath. Gene-targeted disruptants of ChBD-1 and ChBD-15 had no significant differences in invasive growth, pathogenicity, or tolerance to chitinase compared to the wild type. These results suggest that M. oryzae has a mechanism to evade being a substrate for the host chitinase in the first-invaded cell, but neither ChBD-1 nor ChBD-15 contributes to this mechanism.

Ubiquitin ligase EL5 maintains the viability of root meristems by influencing cytokinin-mediated nitrogen effects in rice

Short statement A rice ubiquitin ligase plays a role in preventing root meristematic cell death in the nitrogen-triggered pathway that leads to the production of cytokinin and superoxide.

Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

Rice ubiquitin ligase EL5 prevents root meristematic cell death under high nitrogen conditions and interacts with a cytosolic GAPDH

Root formation in rice transformants overexpressing mutated EL5 (mEL5) was severely inhibited because of meristematic cell death. Cell death was caused by nitrogen sources, particularly nitrate forms, in the culture medium. Nitrite treatment increased the cytokinin contents in roots, but mEL5 contained more cytokinins than non-transformants. Transcriptome profiling showed overlaps between nitrite-responsive genes in non-transformants and genes with altered expression in untreated mEL5. These results indicate that impairment of EL5 function activates nitrogen signaling despite the absence of a nitrogen source. Physical interaction between the EL5 C-terminal region and a cytosolic glyceraldehyde-3-phosphate dehydrogenase, OsGapC2, was demonstrated in vitro and in vivo. Elucidation of the role of glyceraldehyde-3-phosphate dehydrogenase in oxidative cell death in plants is expected in future.