Construction of cDNA Expression Library of Oilseed Rape and Identification of the Interaction Partner of PG, a Virulence Factor from Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is the fungal agent that causes stem rot disease in oilseed rape (Brassica spp.). To overcome plant cell wall during early pathogenesis, the fungus secretes several types of cell wall-degrading enzymes such as polygalacturonases (PGs). The activities of these enzymes determine the pathogenicity/ virulence and are crucial for the colonization on plant tissues. PGs can induce defense reactions in plants unrelated to its enzyme activities. To understand the PG signaling pathway, a PG gene was cloned from S. sclerotiorum by RT-PCR. The encoding region of the PG gene was fused to the yeast GAL4 DNA-binding domain in yeast expression vector PGBKT7 and a rapeseed (B. napus) cDNA expression library was constructed in yeast. PG-interacting proteins were screened in the library using PG as bait by yeast two-hybrid assay. An interacting protein was isolated. Sequencing and BLAST analysis revealed that the protein contained a C2-domain and was predicted to be a Ca2+ binding domain. The protein shared 80.24% identity in amino acids with a C2-domain protein in Arabidopsis with unknown function. The expression levels of the C2-domain protein in floral, leaf, and stem organs of oilseed rape were investigated by semi-quantitative RT-PCR. The C2-domain protein was highly expressed in leaves when inoculated with S. sclerotiorum. The comparisons of the C2-domain proteins identified in oilseed rape with that of other species suggested that aspartic acid residues involving in Ca2+ binding were conserved.