Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

Transcriptome analysis of Clavibacter michiganensis subsp. michiganensis-infected tomatoes: a role of salicylic acid in the host response.

Bacterial canker of tomato (Solanum lycopersicon) caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is an economically important disease. To understand the host defense response to Cmm infection, transcriptome sequences in tomato cotyledons were analyzed by RNA-seq. Overall, 1788 and 540 genes were upregulated and downregulated upon infection, respectively. Gene Ontology enrichment analysis revealed that genes involved in the defense response, phosphorylation, and hormone signaling were over-represented by the infection. Induced expression of defense-associated genes suggested that the tomato response to Cmm showed similarities to common plant disease responses. After infection, many resistance gene analogs (RGAs) were transcriptionally upregulated, including the expressions of some receptor-like kinases (RLKs) involved in pattern-triggered immunity. The expressions of WRKYs, NACs, HSFs, and CBP60s encoding transcription factors (TFs) reported to regulate defense-associated genes were induced after infection with Cmm. Tomato genes orthologous to Arabidopsis EDS1, EDS5/SID1, and PAD4/EDS9, which are causal genes of salicylic acid (SA)-deficient mutants, were upregulated after infection with Cmm. Furthermore, Cmm infection drastically stimulated SA accumulation in tomato cotyledons. Genes involved in the phenylalanine ammonia lyase pathway were upregulated, whereas metabolic enzyme gene expression in the isochorismate synthase pathway remained unchanged. Exogenously applied SA suppressed bacterial growth and induced the expression of WRKYs, suggesting that some Cmm-responsive genes are regulated by SA signaling, and SA signaling activation should improve tomato immunity against Cmm.

Enhanced resistance to fungal and bacterial diseases in tomato and Arabidopsis expressing BSR2 from rice.

KEY MESSAGE: The overexpression of rice BSR2 would offer a simple and effective strategy to protect plants from multiple devastating diseases in tomato and Arabidopsis. Many devastating plant diseases are caused by pathogens possessing a wide host range. Fungal Botrytis cinerea and Rhizoctonia solani, as well as bacterial Pseudomonas syringae and Ralstonia pseudosolanacearum are four such pathogens that infect hundreds of plant species, including agronomically important crops, and cause serious diseases, leading to severe economic losses. However, reports of genes that can confer resistance to broad host-range pathogens via traditional breeding methods are currently limited. We previously reported that Arabidopsis plants overexpressing rice BROAD-SPECTRUM RESISTANCE2 (BSR2/CYP78A15) showed tolerance not only to bacterial P. syringae pv. tomato DC3000 but also to fungal Colletotrichum higginsianum and R. solani. Rice plants overexpressing BSR2 displayed tolerance to two R. solani anastomosis groups. In the present study, first, BSR2-overexpressing (OX) Arabidopsis plants were shown to be additionally tolerant to B. cinerea, R. solani, and R. pseudosolanacearum. Next, tomato 'Micro-Tom' was used as a model to determine whether such tolerance by BSR2 can be introduced into dicot crops to prevent infection from pathogens possessing wide host range. BSR2-OX tomato displayed broad-spectrum disease tolerance to fungal B. cinerea and R. solani, as well as to bacterial P. syringae and R. pseudosolanacearum. Additionally, undesirable traits such as morphological changes were not detected. Thus, BSR2 overexpression can offer a simple and effective strategy to protect crops from multiple destructive diseases.

Low accumulation of chlorogenic acids represses reddening during flesh browning in Japanese peach "Okayama PEH7".

In peaches, fruit flesh browns unattractively after peeling or cutting. A recently developed cultivar, Okayama PEH7, was distinct from other Japanese cultivars, including Okayama PEH8, with respect to its reduced browning potential. Homogenate prepared from Okayama PEH7 flesh had significantly less reddening during the browning reaction. Okayama PEH7 had less soluble phenolic compounds and higher polyphenol oxidase activity than Okayama PEH8. Reduced browning was observed even when phenols prepared from Okayama PEH7 were incubated with crude extract from Okayama PEH8, suggesting that phenols lower the browning potential of Okayama PEH7. In Okayama PEH7, contents of chlorogenic acid and its isomers were about one-tenth compared to Okayama PEH8. Exogenous addition of chlorogenic acid to Okayama PEH7 homogenate increased the browning potential and visibly enhanced reddening. These results indicate that the reduced browning of Okayama PEH7 flesh is due to a defect in chlorogenic acid accumulation.

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.

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.

OsNAC111, a blast disease-responsive transcription factor in rice, positively regulates the expression of defense-related genes.

Plants respond to pathogen attack by transcriptionally regulating defense-related genes via various types of transcription factors. We identified a transcription factor in rice, OsNAC111, belonging to the TERN subgroup of the NAC family that was transcriptionally upregulated after rice blast fungus (Magnaporthe oryzae) inoculation. OsNAC111 was localized in the nucleus of rice cells and had transcriptional activation activity in yeast and rice cells. Transgenic rice plants overexpressing OsNAC111 showed increased resistance to the rice blast fungus. In OsNAC111-overexpressing plants, the expression of several defense-related genes, including pathogenesis-related (PR) genes, was constitutively high compared with the control. These genes all showed blast disease-responsive expression in leaves. Among them, two chitinase genes and one ß-1,3-glucanase gene showed reduced expression in transgenic rice plants in which OsNAC111 function was suppressed by a chimeric repressor (OsNAC111-SRDX). OsNAC111 activated transcription from the promoters of the chitinase and ß-1,3-glucanase genes in rice cells. In addition, brown pigmentation at the infection sites, a defense response of rice cells to the blast fungus, was lowered in OsNAC111-SRDX plants at the early infection stage. These results indicate that OsNAC111 positively regulates the expression of a specific set of PR genes in the disease response and contributes to disease resistance.

Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.

Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus.

WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP-OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein-fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels.

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.