Abstract: 【Objective】The gamma-glutamyl phosphate reductase (GPR) is a key enzyme in fungal proline synthesis pathway. The objective of this study is to silence a GPR-encoded gene SsGPR1 in Sclerotinia sclerotiorum via the RNA interference strategy, research the mycelial growth, sclerotial formation and pathogenicity of the gene-silencing transformants, so as to lay a foundation for revealing the growth, development and pathogenicity of S. sclerotiorum. It also provides important clues for the green prevention and control of Sclerotinia disease.【Method】Homology analysis and phylogenetic tree construction were performed through the BLAST search and MEGA 5.0 software. The real-time RT-PCR was used to detect the expression pattern of SsGPR1 at the different stages of mycelial growth, sclerotial development and germination, and infection processes. The gene silencing vector of SsGPR1 was constructed based on the principle of RNA interference, and the vector was used to transform wild-type strain 1980 by PEG-mediated transformation of protoplasts methods. The gene-silenced strains were identified by real-time RT-PCR. The mycelial morphology, growth rate and sclerotial formation of the gene-silenced strains were observed and the hyphal growth rate under the oxidative stress was measured. The gene-silenced strains were inoculated on Brassica napus leaves and Arabidopsis thaliana plants, and lesion size was observed and measured. The free proline of gene-silenced strains was assayed using the acid ninhydrin method. 【Result】 SsGPR1 of S. sclerotiorum is 1 454 bp in length and encodes 449 amino acids. SsGPR1 protein contains a GPR domain at amino acid H¹⁰-N⁴²⁶. SsGPR1 showed high sequence similarity with Botrytis cinerea BC1G_13183 (95% identities) and Sclerotinia borealis SBOR_2215 protein (94% identities). The three proteins clustered into a small branch according to the result of phylogenetic tree. SsGPR1 showed high expression level during hyphae growth. The expression level was similar during the different development stages of sclerotia, but it significantly decreased compared with that during the hyphal growth period. The expression level of SsGPR1 increased gradually during the pathogenic period, and it reached the highest at 9 h post inoculation. The SsGPR1 silencing vector, pSIGPR1 was transformed into the wild-type strain of S. sclerotiorum, and the expression level of SsGPR1 in different transformants was detected by real-time RT-PCR. The results showed that SiGPR1-104 and SiGPR1-149 were SsGPR1 gene-silenced transformants. When cultured on PDA medium, SsGPR1 gene-silenced strains had no significant difference with wild-type strain on the number and average dry weight of sclerotia, which can germinate to form apothecium. However, the gene-silenced strains produced denser hyphae and showed a significantly reduce in growth rate. The hyphal growth of SsGPR1 gene-silenced strains was inhibited more strongly when cultured on medium containing H₂O₂, indicating that the gene-silenced strains were more sensitive to oxidative stress. SsGPR1 gene-silenced strains led to small lesions on B. napus leaves and A. thaliana plants, indicating that the pathogenicity of the gene-silenced strains was impaired. The content of proline produced by SsGPR1 gene-silenced strains had no significant difference with wild-type strain.【Conclusion】SsGPR1 is related to hyphal growth and mycelial morphology, and involved in the oxidative stress resistance and pathogenicity of S. sclerotiorum.

Key words: Sclerotinia sclerotiorum, proline, gamma-glutamyl phosphate reductase, gene silencing, pathogenicity, oxidative stress