Cloning, Expression And Pathogenicity Analysis Of PG Gene Of Rice Sheath Blight

Rice sheath blight occurring worldwide in rice-producing areas has been one of the most destructive rice diseases. The teleomorph of the causing agent is Thanatephorus cucumeris (Frank) Donk and the anamorph is Rhizoctonia solani Kuhn. This pathogen can infect more than 200 kinds of plant species from 43 genera, such as rice, soybean, maize, wheat, cynodon dactylon and bristlegrass, causing sheath blight and soreshin symptoms. It secrets cell wall degrading enzymes in the process of infecting its hosts. Polygalacturonase (PG) is one of these important enzymes and can facilitate invasion by degrading plant cell walls.In this study, we focused on Rhizoctonia solani YN-7 and cloned three PG genes using PCR and RT-PCR. The gene information is as follows. The full length of PG6934 is 1633 bp long with 6 introns. Its coding sequence is 1311bp long (KP896519), encoding 436 amino acids with a 16 amino acids N-terminal signal peptide. PG9039 is 943 bp long in length with 4 introns and its 720bp coding sequence corresponds to 239 amino acids (KP896520). PG9605 is 1466 bp long in length with 8 introns and its 1038bp coding sequence corresponds to 345 amino acids (KP896521). Bioinformatic analysis showed that all of these three predicted proteins contain conserved functional domains which are characteristic of PG. Four domains of PG6934 are located at 225NTD,248 DD,270SHG and 310 RIK. The domains of PG9039 are located at 58NTD, 80 DD, 101 GHG and 133RIK. The domains of PG9605 are located at 130 ETD,153 DD,175 SHG and 211 RIK. Ten cysteine residues of PG6934 form five disulfide bonds while four Cys of PG9039 result in two bonds and eight Cys of PG9605 form four disulfide bonds. Secondary structure prediction suggested that a-helix, P-sheet and random coils are their basic fundamental structures. Transmembrane analysis predicted that all proteins are mainly transported into extracellular space.Three PG genes were cloned into the plasmid pET-28a and transformed into Escherichia coli BL21 for IPTG-induced prokaryotic expression. However, the activity of polygalacturonase was not detected in either bacterial culture supernatants or bacterial mass treated by ultrasound or several round freezing-thawing. At the same time, inclusion body also did not exhibit any activity when denatured and then renatured. Then, these genes plus one previously-cloned PG gene RsPGl (KP896518) were cloned into the eukaryotic expression vector pPIC9K. Recombinant plasmids were transformed into Pichia pastoris GS115. After induced by methanol, all the supernatants showed polygalacturonase activity. The recombinant yeasts GS-PG6934-7, GS-PG9039-5, GS-PG9605-8 and GS-RsPGl-10 displayed the highest enzyme activity at 101.21U/mL,91.09U/mL,91.09U/m and 267.21U/mL respectively. The rice sheath exhibited obvious necrosis at 48 hours after punching-inoculated with rude enzyme fluid. Also, rice sheath in four-leaf stage displayed typical symptoms when treated with four kinds of rude enzyme fluid. After 24 hours, the amount of reducing sugar showing with the value of OD540 1.387,1.061, 1.053 and 1.664 respectively. Ratio of cell damage was 78.4%,62.6%,55.5% and 90.0% respectively. Real-time PCR data showed that these four genes were up-regulated during the rice infection of the pathogen. The expression level peaked at 48 hours after inoculation and the expression of different genes was varying significantly. These results indicated that all the genes may play roles in the pathogenicity of the pathogen on the host plants.