Gene Function Of BkLiP1 And Identification Of Botryosphaeria Causing Pear Ring Rot In China

Pear ring rot is an important disease that can damage branches,leaves,and fruits,causing branch and leaf death and fruit rot and seriously threatening the development of the pear industry in the main pear-producing areas in China.At present,several Botryosphaeria fungi are responsible for this disease,but their virulence mechanisms are scarcely addressed.In this study,branches showing typical symptoms of pear ring rot were collected from different pear-producing areas in China for fungus isolation and identification,to identify the dominant species.Combined with the symptoms induced by Botryosphaeria,the gene encoding lignin peroxidase（LiP）in the dominant species is proposed,with gene knock-out,complementation,and overexpression,to analyze the biological function of theLiP.This study was aimed at providing a new experimental basis for the analysis of molecular pathogenesis of the Botryosphaeria.The results of this research are as follows:1.Identification of Botryosphaeria causing pear ring rot in China.In total,232 branch samples showing typical symptoms of pear ring rot were collected from 16 provinces and cities（Anhui,Beijing,Chongqing,Fujian,Hebei,Henan,Hubei,Jiangsu,Jiangxi,Liaoning,Sichuan,Shandong,Shaanxi,Shanxi,Yunnan,and Zhejiang）,including 124 stem wart samples and 108 stem canker samples.A total of 341 Botryosphaeria isolates were acquired by isolation and purification using a tissue isolated method,of which 174 isolates were acquired from stem wart samples and 167 isolates were obtained from stem canker samples.Morphological characteristics and phylogenetic analysis with SSU,ITS,and EF1-αsequences for all the isolates,the results showed that 302 isolates belonged to B.kuwatsukai,which could be isolated from branch samples showing stem wart or stem canker symptoms,and distributed on cultivated pears in the south and north of China.And 39 isolates were B.dothidea,which was only isolated from stem canker samples,mainly distributed in the pear-producing areas in the north of China.In addition,12 representative strains of B.kuwatsukai and B.dothidea were selected respectively for pathogenic test on branches from 1-year-old pear saplings in vivo and in vitro.The results showed that B.kuwatsukai had significantly more virulence on pear branches than B.dothidea,and could produce stem wart and stem canker,while B.dothidea only caused stem canker.Our results indicate that B.kuwatsukai is the dominant species causing pear ring rot in the main producing areas in China,which could produce two kinds of symptoms.2.The sequence analysis and biological function of aLiP gene in B.kuwatsukai.LiP is the main lignin-degrading enzyme in the process of lignin biodegradation,which has been found in white-rot fungi.In this study,seven genes encoding lignin peroxidases（BkLiPs）were found using bioinformatics methods from B.kuwatsukai strain SZ2l62 with higher virulence.Gene sequence analysis showed that the open reading frame of BkLiP1 is1116 bp encoding a 371-amino-acid（aa）protein with a predicted N-terminal signal peptide（1-21 aa）.The full-length of BkLiP2 is 1048 bp including two introns,and the encoded protein of BkLiP2 has 312 aa carrying predicted a signal peptide（1-19 aa）at the N-terminus.The phylogenetic analysis ofLiP paralogs showed that BkLiP1-BkLiP4 was a peroxidase protein-containing signal peptide,which was very conservative in ascomycetes;BkLiP5-BkLiP7 was a peroxidase protein without signal peptide.The RT-q PCR analysis suggested that BkLiP1 and BkLiP2 genes in B.kuwatsukai were up-regulated in the infected pear tissues.In addition,BkLiP1 was a secreted protein as confirmed in yeast.Based on the BkLiP1 gene knock-out,complementation,and overexpression analysis,the mycelial growth rate of BkLiP1 gene knock-out and lacking signal peptides mutants were reduced,while the conidia induction and mycelial dry weight were not affected,but the virulence were both weakened.Compared with the wild type,the virulence of the gene complementation and overexpression transformants were recovered and significantly enhanced respectively.The virulence comparison among the six cysteine mutants of BkLiP1 indicated that only the c BkLiP1^C92A transformant was less virulence than the wild type.These results confirm that the BkLiP1 play an important role in the virulence of B.kuwatsukai.Moreover,the results of subcellular localization showed that the BkLiP1 was located in the apoplast space of Nicotiana benthamiana cells.And the transient expression of BkLiP1 could cause necrosis,reactive oxygen species burst,callosum accumulation,and immunity-related genes up-regulated in the leaves of N.benthamiana.Our results suggest that BkLiP1 secreted by B.kuwatsukai is a MAMP,and triggers the defense response of plants.In this study,it is the first time to confirm that B.kuwatsukai is the dominant species causing pear ring rot in main producing areas in China,which caused two symptoms with stem wart and stem canker.The lignin peroxidase gene BkLiP1 encoded by B.kuwatsukai plays an important role in the virulence of pear,and BkLiP1 protein can also trigger the immune response in N.benthamiana.The results provide new evidence to explore the molecular pathogenesis of B.kuwatsukai,which is significant for pear ring rot prevention and control in the production area.