Functional Characterization Of A β-1,3-Glucanase Gene Osg1 In Rice

β-1,3-glucanases (glucan endo-1,3-β-glucosidases, E.C.3.2.1.39, p-1,3-GLUs) catalyze the hydrolysis ofβ-1,3-glucan, which is a polymer ofβ-1,3-linked glucose residues.β-1,3-GLUs have been found in plants, yeasts, actinomycetes,bacteria,fungi, insects, and fish. P-1,3-glucan is a major structural component of the cell walls of many pathogenic fungi.β-1,3-glucan (called callose in plants) is also a cell-wall component of certain cell types during specific developmental stages of plants. Plantβ-1,3-GLUs are pathogenesis-related proteins and they are classified as members of the PR-2 family. Syntheses of these enzymes can be induced by pathogens or other stimuli. Because the substrate forβ-1,3-GLUs exists in plants, these enzymes may be involved in various physiological and developmental processes, such as cell elongation, cell division, fruit ripening (cell wall degradation leading to fruit softening), fertilization, pollen germination and tube growth, bud dormancy (removal of phloem callose), microsporogenesis (dissolution of pollen tetrads into free microspores), somatic embryogenesis, seed germination and flower formation.In rice, more than 10 genes encoding putativeβ-1,3-GLUs have been isolated and sequenced. Only limited information is presently available on regulation of gene expression and function of the proteins encoded by individualβ-1,3-GLU genes. In the study here, we analysis the nucleotide and deduced amino acid sequence of aβ-1,3-GLU gene Osgl isolated from the rice anther. The phylogenic tree of the deduced Osgl protein with several riceβ-1,3-GLUs shows that Osgl belongs to the monocotyledon subgroup A, which is comprised of defense-relatedβ-1,3-GLUs and is the largest glucanase subgroup in monocotyledons. The 5'-upstream region of Osgl contained four copies of the pollen-specific cis-acting elements POLLEN1LELAT52, seven copies of the "GTGA motif" found within the promoter of the tobacco late pollen gene g10 and a "Q(quantitative)-element" found in a pollen-specific maize gene ZM13 promoter. These pollen-specific and quantitative elements suggest that Osgl involved in the development of the pollen.We also report a detailed functional characterization of Osgl. Osgl is expressed throughout the plant, with highest expression in leaf blades and florets. Then we examined Osgl activity using transgenic plants carrying the fusion constructs of the Osgl promoter region and the GUS reporter gene. We found that Osgl was expressed mainly in the tapetal cells in the anthers. To elucidate the role of Osgl in rice growth and development, we constitutively suppressed the expression of Osgl gene in transgenic rice. The RNA interference (dsRNAi) construct (Osgl-RI) of Osgl were developed under the control of the maize ubiquitin 1 (Ubil) promoter. Transgenic plants were generated by introducing the constructs into the japonica rice variety H1493 by Agrobacterium-mediated transformation. We demonstrated that silencing of Osgl by RNA interference (RNAi) resulted in pleiotropic developmental abnormalities including retarded and stunted growth and male sterility in rice, suggesting that Osgl plays critical roles in plant growth and development.Osgl-RI plants are male-sterile but female-fertile. Pollen mother cells (PMCs) of Osgl-RI plants appeared to be normal prior to the young microspore stage. However, at the stage of early young microspore, normal callose degradation was delayed in PMCs of Osgl-RI plants, consequently the release of the young microspore into the anther locules was delayed and the PMCs began to degenerate at the early young microspore stage. These results demonstrated here provide the direct evidence that Osgl is essential for callose degradation during pollen development.Hao et al. (2008) obtained indications that Osgl was strongly induced in susceptible plants and the deposited callose was decomposed thereby facilitating the brown planthopper (Nilaparvata lugens Stal; BPH) continued feeding from the phloem. However, this gene was induced much more weakly in resistant plants. Thus, differential expression of Osgl should account for between-plant differences in resistance levels. Thus, we recorded in detail the feeding behavior of BPH on rice plants in real time employing the electronic penetration graph (EPG) analysis. The EPG data showed that the duration of non-probing and penetration was significantly longer and the duration of phloem ingestion was clearly shorter on the Osgl-RI plants, compared with the wild-type plants. These results demonstrate that Osgl confers an antibiotic resistance that reduces the feeding of the BPH insects.