蔡 晃植

During development, plants are continuously confronted with diverse pathogens. However, plants are resistant to most microbes and rely entirely on plant immune responses for their defense. Plants have evolved a multi-layered defense system that can be activated upon pathogen invasion. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, induces plant immune responses including H2O2 generation, while flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H2O2 generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferasedeficient mutants showed that 1,600 Da and 2,150 Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (178Ser, 183Ser, 212Ser and 351Thr) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, 178Ser/Ala and 183Ser/Ala K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at 178Ser and 183Ser in K1 flagellin prevent epitope recognition in rice.

We next examined additional factors, distinct from flagellin, that act as inducers of immune responses in rice. Cell extracts isolated from flagellin-deficient N1141 (Df la1141) still induced PTI responses, suggesting that Df la1141 possesses an additional PAMP distinct from flagellin. Here, we show that elongation factor Tu (EF-Tu), one of the most abundant bacterial proteins, act as a PAMP in rice and causes several PTI responses. In Brassicaceae species, EF-Tu and an N-acetylated peptide comprising the first 18 amino acids of the N-terminus, termed elf18, are fully active as inducers of PTI responses. By contrast, elf18 did not cause any immune responses in rice, whereas an EF-Tu middle region comprising Lys176 to Gly225, termed EFa50, is fully active as a PAMP in rice. In the leaves of rice plants, EF-Tu induced H2O2 generation and callose deposition, and also triggered resistance to co-infection with pathogenic bacteria. Taken together, these data demonstrate that rice recognizes EFa50, which is distinct from elf18, and that this epitope induces PTI responses.

To clarify the induction mechanism of Hypersensitive cell death (HR), we identified several genes that are upregulated during HR cell death using PCR subtraction analysis and microarray analysis. Among the identified genes, the OsNAC4 gene encoding a plant-specific transcription factor was identified in both analyses. Overexpression of OsNAC4 leads to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes. In OsNAC4 knock-down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. After induction by an avirulent pathogen recognition signal, OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner. To understand the molecular mechanisms by which OsNAC4 mediates cell death, we compared global gene expression in the OsNAC4-RNAi cell line with a control line using a 22,000 elements oligonucleotide microarray. A microarray analysis demonstrated that 139 genes including OsHSP90 and IREN, encoding a Ca2+-dependent nuclease, were expressed differently between the OsNAC4-RNAi and control lines during HR cell death. When OsHSP90 was transiently expressed in rice cells, loss of plasma membrane integrity was rapidly induced, while no nuclear DNA fragmentation was observed. We next cloned the full-length IREN cDNA into a vector under the control of the ubiquitin promoter, and introduced this construct into rice cells. After TUNEL staining, all of the IREN-expressing cultured rice cells exhibited fluorescein-derived bright-green fluorescence of their nuclei, suggesting that IREN overexpression causes rapid DNA fragmentation in nuclei. While no cell death induction was detected when IREN was transiently expressed in rice protoplasts. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways.