Screening of pathogenicity defective mutants and analysis of flanking sequence in Valsa pyri

Abstract:【Objective】Pear is one of the most important fruits worldwide and China is a major production area. However, certain microbial pathogens and insects pose a great threat to pear production. Among pear tree diseases, pear Valsa canker disease caused by Valsa pyri is one of the most serious threats to pear growth in East Asia including China. V. pyri can invade host tissue wounded by injury in the bark. Following V. pyri infection, pear trees exhibit reddish-brown, water-soaked, softened barks, and even plant death in some cases, resulting in severe economic losses. In the important pear planting regions of China, like Northwest China, pear Valsa canker disease has become more and more common and destructive in recent years, which greatly affects the development of pear industry. However, it is difficult to control the disease by chemical application, because V. pyri can penetrate systemically in xy- lem and phloem tissues and most active compounds of fungicides are not able to protect the internal phases of the tree trunk. In addition, this pathogen can infect the host plant at any time of the year. Therefore, the functional characterization of virulence genes and pathogenic mechanism of V. pyri are urgently needed, which would help us to make comprehensive prevention and controlling strategy. How- ever, most studies on V. pyri conducted to date have focused on the identification of this pathogen, inves- tigation of disease regularity and fungicide selection, rather than on the actual basis for its pathogenesis. The aim of this study is to identify pathogenicity defective mutants from a T-DNA insertion mutant li- brary of V. pyri and to analyze pathogenicity-related genes, which are expected to provide a better under- standing of the pathogenic mechanism of V. pyri.【Methods】We used an approach to the Agrobacterium tumefaciens-mediated transformation (ATMT) of V. pyri with a plasmid vector encoding the green fluo- rescent protein (GFP) and hygromycin resistance (Hyg) genes. Transformants were screened by placing on PDA plates containing 50 mg · L- 1 hygromycin B. The hygromycin B- resistant transformants were then used for genomic DNA extraction. After that, DNA was utilized as a template together with the specific primers by PCR analysis in order to test for successful transformation. Meanwhile, GFP expression of transformants was evaluated via laser scanning confocal microscope to further identify the positive transformants. After identification, the positive transformants were tested for their virulence using a pear fruit infection assay and the pathogenicity defective mutants were further confirmed by using pear branches as inoculation material. The morphology of pathogenicity defective mutants including colony growth rate was also assessed by culturing these fungi for 7 days on hygromycin B-free PDA. T-DNA insertion copy number of pathogenicity defective mutants was identified by using Southern blot assay. DNA sequences flanking the T-DNA insertional sites of each pathogenicity defective mutant were amplified by thermal asymmetric interlaced PCR (TAIL-PCR). The obtained flanking sequences were used to analyze by comparison with genome sequences of V. pyri【. Results】In total, 504 hygromycin B-resistant transformants were obtained by ATMT of V. pyri. Eight randomly chosen transformants produced the expected 590 bp GFP fragment following PCR amplification, while WT colonies exhibited no such band. The result of GFP fluorescence detection revealed that the hyphae of hygromycin B-resistant colo- nies exhibited robust and uniform GFP signal, whereas WT colonies did not exhibit any GFP signal, which indicated that these hygromycin B-resistant colonies were positive transformants. The result of pathogenicity test showed that 4 transformants (T8, T12, T43 and T75) from 250 exhibited smaller dis- ease lesion on pear fruits after inoculation, while the lesion diameter was about 3.2 cm after inoculation with WT strain. This result was consistent with that using pear branches as inoculation material. These results indicated that transformants T8, T12, T43 and T75 were pathogenicity defective mutants. The morphology of the four transformants was assessed, from which T43 showed significantly different by comparing with the WT strain. The colony growth rate of T43 mutant was greatly reduced by 9.2% and distinct nicks were on the edge of the colony. The other three mutants showed similar morphology with the WT strain. Southern bolt result revealed that three mutants (T8, T43 and T75) contained one T-DNA insertion, while T12 contained more T-DNA insertions. The specific brand was obtained from mutant T8, T43 and T75 by isolating flanking sequences of T-DNA with TAIL-PCR method, while no specific brand was gotten from T12 mutant. Then the insertion position of T-DNA in three mutants was identi- fied and analyzed by comparison with genome sequences of V. pyri. The BLAST result showed that the T-DNA in T43 and T75 was inserted at the coding region of Zds1 gene and putative glutamate synthase gene, respectively, while T- DNA in T8 was inserted at terminator region of MSF domain containing gene. Theses insertions may affect the indicated genes expression, which caused the reduction of patho- genicity in mutants.【Conclusion】A library of T-DNA insertion mutants mediated by Agrobacterium tu- mefaciens was constructed. Four pathogenicity defective mutants were obtained by screening and the flanking sequences of T-DNA insertional sites of each mutant. This laid a foundation for the further in- vestigation on the pathogenic mechanism of V. pyri.