WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89-eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses.

The WRKY transcription factor (TF) gene family expanded greatly in the evolutionary process from green algae to flowering plants through whole genome, segmental, and tandem duplications. Genomic sequences from diverse plant species provide valuable information about the origin and evolution of WRKY domain-containing proteins. Accumulating data indicate that WRKY TFs bind W-box and/or other cis-elements, revealing the specificity of ciselement recognition based on the structure of the WRKY protein as well as the nucleotides adjacent to the core sequences. Physical interactions among WRKY proteins or between WRKY proteins and other regulatory factors afford important insights into the regulation of this TF family. WRKY proteins are essential players in the kinase signaling network. The interaction of plant resistance (R) proteins with WRKY TFs and the existence of unusual chimeric R-WRKY proteins suggest diversity in signaling pathways for rapid immune responses. In this review, we focus mainly on progress in understanding the function of WRKY TFs in response to biotic stresses and focus on their multiple roles in Arabidopsis and rice plants.

Osmotin promoter binding protein 1 (OPBP1), an AP2/EREBP-like transcription factor of tobacco (Nicotiana tabacum), was isolated using a yeast one-hybrid system. RNA gel blot analysis indicated that expression of the OPBP1 gene was induced by elicitor cryptogein, NaCl, ethephon, methyl jasmonate, as well as cycloheximide. Transient expression analysis using an OPBP1-eGFP fusion gene in onion epidermal cells revealed that the OPBP1 protein was targeted to the nuclear. Further, electrophoretic mobility shift assays demonstrated that the recombinant OPBP1 protein could bind to an oligonucleotide containing the GCC-box cis element. Transgenic tobacco plants with an over expression of the OPBP1 gene accumulated high levels of PR-1a and PR-5d genes and exhibited enhanced resistance to infection by Pseudomonas syringae pv tabaci and Phytophthora parasitica var nicotianae pathogens. They also exhibited increased tolerance to salt stress. These results suggest that OPBP1 might be a transcriptional regulator capable of regulating expression in sets of stress-related genes.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.