苹果轮纹病菌(Botryosphaeria dothidea)果胶裂解酶基因Bdpl1的致病功能分析
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摘要
由葡萄座腔菌(Botryosphaeria dothidea)引起的苹果轮纹病是影响我国苹果安全生产的重大病害之一。因此,本研究基于受苹果轮纹菌侵染的苹果组织中果胶裂解酶基因Bdpl1表达上调这一现象,通过Split-marker PCR技术构建Bdpl1基因敲除载体,并通过PEG介导的原生质体转化获得转化子,经常规PCR和qRT-PCR对所获得的转化子进行筛选,成功获得1个Bdpl1基因缺失阳性突变子。该转化子在PDA上的培养基性状与野生型没有明显差异,但在果胶培养基上菌落直径明显小于野生型。其胞外果胶酶活相比野生型明显下降,但在离体"早富"苹果枝条上的致病力并没有明显的下降。通过qRT-PCR技术发现在基因Bdpl1敲除后,其家族内有3个基因在病菌侵染过程中相比野生型明显上调表达(>3倍)。这些现象表明果胶裂解酶基因Bdpl1与病原菌的营养生长过程关系不大,但其参与对寄主果胶类物质的降解。Bdpl1基因对轮纹病菌致病力的影响较小,有可能是Bdpl1基因敲除后,该基因家族内其他基因的上调表达补偿了Bdpl1基因的功能。
Apple ring rot caused by Botryosphaeria dothidea is one of the devastating diseases in China,which has become a major threat to apple production. In preliminary experiments,we found the expression of pectin lyase gene( Bdpl1) was obviously up-regulated in diseased tissues than that in fungal mycelia. Therefore,the Bdpl1 was considered as a virulence gene involved in the pathogenic processes. In order to analyze its pathogenic function,the knockout vector of Bdpl1 was constructed by the split-marker method and the transformants were obtained through PEG-mediated protoplast transformation. One mutant,△Bdpl1-3,was confirmed with four pairs of primers. Compared with the wild-type strain,the △Bdpl1-3 morphology had no significant differences on PDA medium,whereas the colony diameter on pectin medium was much smaller than the wild-type one. The extracellular pectinase activity of the △Bdpl1-3 isolate was lower than the wild type. Nevertheless,pathogenicity of the △Bdpl1-3 isolate on excised one-year-old‘Fuji'apple twigs had no significant differences with the wildtype strain.Relative expression levels of several other pectin lyase( PL) family genes in the △Bdpl1-3 and wildtype strains were detected by qRT-PCR. The results showed that the expression of three other PL family genes were up-regulated( over three folds than the wild-type strain). These data indicated that the Bdpl1 had no obviously effects on fungal growth and pathogenicity,but it had significant acceleration to degrade pectin. In the △Bdpl1-3 strain,other PL family genes might compensate the functions of Bdpl1 by up-regulated expression.
Apple ring rot caused by Botryosphaeria dothidea is one of the devastating diseases in China,which has become a major threat to apple production. In preliminary experiments,we found the expression of pectin lyase gene( Bdpl1) was obviously up-regulated in diseased tissues than that in fungal mycelia. Therefore,the Bdpl1 was considered as a virulence gene involved in the pathogenic processes. In order to analyze its pathogenic function,the knockout vector of Bdpl1 was constructed by the split-marker method and the transformants were obtained through PEG-mediated protoplast transformation. One mutant,△Bdpl1-3,was confirmed with four pairs of primers. Compared with the wild-type strain,the △Bdpl1-3 morphology had no significant differences on PDA medium,whereas the colony diameter on pectin medium was much smaller than the wild-type one. The extracellular pectinase activity of the △Bdpl1-3 isolate was lower than the wild type. Nevertheless,pathogenicity of the △Bdpl1-3 isolate on excised one-year-old‘Fuji'apple twigs had no significant differences with the wildtype strain.Relative expression levels of several other pectin lyase( PL) family genes in the △Bdpl1-3 and wildtype strains were detected by qRT-PCR. The results showed that the expression of three other PL family genes were up-regulated( over three folds than the wild-type strain). These data indicated that the Bdpl1 had no obviously effects on fungal growth and pathogenicity,but it had significant acceleration to degrade pectin. In the △Bdpl1-3 strain,other PL family genes might compensate the functions of Bdpl1 by up-regulated expression.
引文
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[21]Chen L,Sun G W,Wang H K,et al.Protoplast preparation and gfp transformation of Botryosphaeria dothidea(in Chinese)[J].Scientia Silvae Sinicae(林业科学),2014,50(6):131-137.
[22]Zang R,Huang L L,Kang Z S,et al.Biological characteristics and pathogenicity of different isolates of Cytospora spp.Isolated from apple trees in Shanxi province(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2007,37(4):343-351.
[23]Xu C J.Pathogenic function of three pectinase genes in Valsa mali(in Chinese)[D].Yangling:Northw est A&F University(杨凌:西北农林科技大学),2016.
[24]Yao S H,Jia L J,Zhang X W,et al.Analysis of a pectate lyase PelA in Fusarium graminearum(in Chinese)[J].Plant Physiology Journal(植物生理学报),2014,50(3):243-252.
[25]Fu L.Cloning and functional analysis of PEL(Pectate Lyase)gene from Phytophthora capsici(in Chinese)[D].Tai’an:Shandong Agricultural University(泰安:山东农业大学),2012.
[26]Bendaniel B H,Barzvi D,Tsror L L.Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines[J].Molecular Plant Pathology,2012,13(2):187-197.
[27]Xu C J,Sun Y C,Wu Y X,et al.Pathogenic function of pectate lyase gene Vmpl4 of Valsa mali in apple(in Chinese)[J].Journal of Fruit Science(果树学报),2017,34(1):19-25.
[2]Han Q M,Gao X N,Wang J Q,et al.Cytological and histological studies of the interaction between Botryosphaeria dothidea and apple tw igs[J].Scientia Horticulturae,2016,202(2):142-149.
[3]Kim K W,Park E W,Kim Y H,et al.Latency-and defense-related ultrastructural characteristics of apple fruit tissues infected with Botryosphaeria dothidea[J].Phytopathology,2001,91(2):165-172.
[4]Kang L,Hao H M,Yang Z Y,et al.The advances in the research of apple ring rot(in Chinese)[J].Chinese Agricultural Science Bulletin(中国农学通报),2009,25(9):188-191.
[5]Tang W,Ding Z,Zhou Z Q,et al.Phylogenetic and pathogenic analyses show that the causal agent of apple ring rot in China is Botryosphaeria dothidea[J].Plant Disease,2012,96(4):486-496.
[6]Xiao L,Zhang C X,Zong Z R,et al.Proteomic analysis of pathogen-responsive proteins from the chloroplast of apple leaves induced by Botryosphaeria dothidea(in Chinese)[J].Journal of Fruit Science(果树学报),2016,33(11):1357-1366.
[7]Zhang C X,Tian Y,Zhang L Y,et al.Proteomic analysis of pathogen-responsive proteins from the bark of apple branches induced by Botryosphaeria dothidea(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2015,45(3):280-287.
[8]Zhao N,Zhang W W,Gao X N,et al.Phylogenetic relationships of Botryosphaeria dothidea and its allies in China(in Chinese)[J].Journal of Northw est A&F University(Natural Science Edition)[西北农林科技大学学报(自然科学版)],2011,39(10):123-133.
[9]Sun X Y.Pathogen identification and diversity of apple white rot in Shaanxi province(in Chinese)[D].Yangling:Northw est A&F University(杨凌:西北农林科技大学),2010.
[10]Xu C,Wang C S,Ju L L,et al.Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China[J].Fungal Diversity,2015,71(1):215-231.
[11]An J D,Guo L Y,Zhu X Q,et al.Susceptibility of Botryosphaeria dothidea to tebuconazole and thiophanate-methy(in Chinese)[J].Plant Protection(植物保护),2016,42(2):187-191.
[12]Liu Z,Zhang H L,Xie X B,et al.Induction of defensive response against Botryosphaeria berengriana f.sp.piricola by four elicitors in harvested Fuji apple fruit(in Chinese)[J].Journal of Fruit Science(果树学报),2009,26(5):666-670.
[13]Wang Y Z,Zhang W,Liu B Y,et al.Research on resistance and geographical distribution of Botryosphaeria dothidea from apple to Tebuconazole in Shandong province(in Chinese)[J].Journal of Fruit Science(果树学报),2010,27(6):961-964.
[14]Yang W H,Liu K Q.Resistance detection of Botryosphaeria berengeriana f.sp piricola to carbendazim and thiophanate-methyl(in Chinese)[J].Acta Phytophylacica Sinica(植物保护学报),2002,29(2):191-192.
[15]Alghisi P,Favaron F.Pectin-degrading enzymes and plant-parasite interactions[J].European Journal of Plant Pathology,1995,101:365-375.
[16]Dean R A,Timberlake W E.Regulation of the Aspergillus nidulans pectate lyase gene(pelA)[J].Plant Cell,1989,1(3):275-284.
[17]Yakoby N,Benomoualem D,Keen N T,et al.Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction[J].Molecular Plant Microbe Interactions,2001,14(8):988-995.
[18]Cho Y,Jang M,Srivastava A,et al.A Pectate lyasecoding gene abundantly expressed during early stages of infection is required for full virulence in Alternaria brassicicola[J].PLoS One,2015,10(5):1-18.
[19]Li G X,Shen Y B,Gao Y Y,et al.Study on the infection mechanism of apple ring rot disease(in Chinese)[J].Journal of Fruit Science(果树学报),2007,24(1):16-20.
[20]Li G X,Shen Y B,Gao Y M,et al.Infection process of Botryosphaeria dothidea in apple fruit(in Chinese)[J].Journal of Fruit Science(果树学报),2006,23(1):69-72.
[21]Chen L,Sun G W,Wang H K,et al.Protoplast preparation and gfp transformation of Botryosphaeria dothidea(in Chinese)[J].Scientia Silvae Sinicae(林业科学),2014,50(6):131-137.
[22]Zang R,Huang L L,Kang Z S,et al.Biological characteristics and pathogenicity of different isolates of Cytospora spp.Isolated from apple trees in Shanxi province(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2007,37(4):343-351.
[23]Xu C J.Pathogenic function of three pectinase genes in Valsa mali(in Chinese)[D].Yangling:Northw est A&F University(杨凌:西北农林科技大学),2016.
[24]Yao S H,Jia L J,Zhang X W,et al.Analysis of a pectate lyase PelA in Fusarium graminearum(in Chinese)[J].Plant Physiology Journal(植物生理学报),2014,50(3):243-252.
[25]Fu L.Cloning and functional analysis of PEL(Pectate Lyase)gene from Phytophthora capsici(in Chinese)[D].Tai’an:Shandong Agricultural University(泰安:山东农业大学),2012.
[26]Bendaniel B H,Barzvi D,Tsror L L.Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines[J].Molecular Plant Pathology,2012,13(2):187-197.
[27]Xu C J,Sun Y C,Wu Y X,et al.Pathogenic function of pectate lyase gene Vmpl4 of Valsa mali in apple(in Chinese)[J].Journal of Fruit Science(果树学报),2017,34(1):19-25.