Expression of RSOsPR10 in rice roots is antagonistically regulated by jasmonate/ethylene and salicylic acid via the activator OsERF87 and the repressor OsWRKY76, respectively.

Plant roots play important roles in absorbing water and nutrients, and in tolerance against environmental stresses. Previously, we identified a rice root-specific pathogenesis-related protein (RSOsPR10) induced by drought, salt, and wounding. RSOsPR10 expression is strongly induced by jasmonate (JA)/ethylene (ET), but suppressed by salicylic acid (SA). Here, we analyzed the promoter activity of RSOsPR10. Analyses of transgenic rice lines harboring different-length promoter::ß-glucuronidase (GUS) constructs showed that the 3-kb promoter region is indispensable for JA/ET induction, SA repression, and root-specific expression. In the JA-treated 3K-promoter::GUS line, GUS activity was mainly observed at lateral root primordia. Transient expression in roots using a dual luciferase (LUC) assay with different-length promoter::LUC constructs demonstrated that the novel transcription factor OsERF87 induced 3K-promoter::LUC expression through binding to GCC-cis elements. In contrast, the SA-inducible OsWRKY76 transcription factor strongly repressed the JA-inducible and OsERF87-dependent expression of RSOsPR10. RSOsPR10 was expressed at lower levels in OsWRKY76-overexpressing rice, but at higher levels in OsWRKY76-knockout rice, compared with wild type. These results show that two transcription factors, OsERF87 and OsWRKY76, antagonistically regulate RSOsPR10 expression through binding to the same promoter. This mechanism represents a fine-tuning system to sense the balance between JA/ET and SA signaling in plants under environmental stress.

Loss of chloroplast-localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection.

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine-specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62- and atpp2c26-deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis-related protein, chaperonin-60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two-dimensional isoelectric focusing sodium dodecylsulfate-polyacrylamide gel electrophoresis (2D-IDF-SDS-PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.

Surface α-1,3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants.

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.

Elderberry bark lectins evolved to recognize Neu5Ac alpha2,6Gal/GalNAc sequence from a Gal/GalNAc binding lectin through the substitution of amino-acid residues critical for the binding to sialic acid.

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.

Characterization and cDNA cloning of monomeric lectins that correspond to the B-Chain of a type 2 ribosome-inactivating protein from the bark of Japanese elderberry (Sambucus sieboldiana).

Two monomeric lectins, SSA-b-3 and SSA-b-4, were purified from the bark tissue of Japanese elderberry, Sambucus sieboldiana. SDS-PAGE of the purified lectins showed the presence of single bands of 35 and 33 kDa for SSA-b-3 and SSA-b-4, respectively, irrespective of the presence of reducing agent. MS analysis as well as gel filtration of these lectins indicated that they exist mostly as monomeric lectins. Analysis of the N-terminal amino acid sequences of SSA-b-3 and SSA-b-4 yielded an identical sequence, indicating their close structural relationship. Four cDNA clones with extensive homology were obtained from the bark cDNA library and indicated to encode SSA-b-3 or SSA-b-4 from the comparison with the N-terminal sequences of these lectins. These clones were classified into two groups, three for SSA-b-3 and one for SSA-b-4, based on the predicted isoelectric points. The amino acid sequences of the encoded polypeptides were almost identical with the B-chain of a type 2 ribosome-inactivating protein from the same bark tissue, sieboldin-b, except for the absence of a small peptide containing a cystein residue, which is critical for the heteromeric dimerization with an A-subunit. Carbohydrate binding specificity and biological activity of these lectins are also reported.

Cloning and characterization of cDNAs for the jasmonic acid-responsive Genes RRJ1 and RRJ2 in suspension-cultured rice cells.

Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine gamma-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.