Functional Analysis Of Conidiation Associated Gene CmPrat Of Coniothyrium Minitans

Coniothyrium minitans is an important biocontrol agent of rapeseed stem rot caused by Scleroinia sclerotiorum, which is widely distributed in the world. In this paper, a conidiation deficient mutant ZS-1T21882 of C. minitans was studied, and cmPrat gene, encoding the enzyme phosphoribosylamidotransferase (PRAT; EC 2.4.2.14) was cloned. The function of the gene in conidiation of C. minitans was analyzed using gene disrupion and gene complemental techique. The results were as followed:The colony morphology of mutant ZS-1T21882 was significantly different from the wild type ZS-1 on potato dextrose agar (PDA), including slow growth, close to the surface of medium and no aerial mycelium. The mutant lost the capacity to synthesize anti-fungal, anti-bacterial substances, and lost the ability to produce conidia. But dual cultured with S. sclerotiorum, ZS-1T21882 could restore the conidiation ability partly, producing black conidia in the confrontation regions and on the sclerotia of S. sclerotiorum.Based on cDNA sequence obtained previously, a 6423 bp fragment of the genomic DNA and flanking sequence was obtained by Inverse-PCR amplification. Sequence analysis revealed that the T-DNA insertion damaged a possible gene, whose nucleotide sequence contains a 1875 bp open reading frame (ORF) and two introns (70 bp and 53 bp). The ORF encoded putatively a protein with 584 amino acids, phosphoribosylamidotransferase (PRAT, EC 2.4.2.14). Here, the gene in C. minitans named cmPrat, and the protein CMPRAT. The amino acid sequence of CMPRAT showed 82% and 65% identity with phosphoribosylamidotransferase from Pyrenophora tritici-repentis Pt-1C-BFP and Aspergillus fumigatus Af293.The cmPrat gene in wild type ZS-1 was knock-out with Agrobactirium tumfacience mediated-transformation (ATMT). The phenotype of three knock-out transformants could not be distinguished from mutant ZS-1T21882, and exogenous IMP could recover their conidiation. Southern blot analysis clearly showed that the internal part of the gene was deleted in the mutants, as expected. Then, the gene was transformed into the knock-out mutant KO363 to complement the gene function, and the conidiation of three complementary mutants have been recovered at different degrees. Further results showed that AMP and cAMP could restore the conidiation when amended into PDA medium, but GMP or cGMP could not. The amending experiments indicated that phosphoribosylamidotransferase limits the conidiation of C. minitans via the cAMP signaling pathway.The interaction of cAMP-dependent signaling pathway when C. minitans parasitize S. sclerotiorum was studied preliminarily. The adenylate cyclase (SAC) of S. sclerotiorum was silenced using the RNAi technique, further results showed that conidiation of ZS-1T21882 could not be restored or restored slightly when dual cultured with the silence mutants of Sac. Thus, our study further suggested that in the process of parasitic of S. sclerotiorum, C. minitans absorbed cAMP from the host and applied to their own cAMP signaling pathway.This paper focused on that phosphoribosylamidotransferase (CMPRAT) limits the conidiation of C. minitans via the cAMP signaling pathway, and supplied some ideas about the interaction of cAMP-dependent signaling pathway between C. minitans and S. sclerotiorum during parasitic affairs.