Screening And Analysis Of Genes Associated With Soft Rot In Zantedeschia Spp.

Zantedeschia spp.,commonly known as calla lilies,is one of the most popular ornamental plants in the world due to its unique shape and diverse colors.However,calla lilies are often affected by soft rot disease caused by Pectobacterium carotovora subsp.carotovorum(Pcc)during production,and infected calla lilies can rapidly rot and die.Soft rot disease is the most serious disease that causes tissue necrosis and damages the growth and development of calla lilies.With the development of molecular techniques,research on soft rot disease-resistant genes and susceptible genes has led to the discovery and utilization of soft rot disease-related genes in calla lilies as an important method for preventing and controlling soft rot disease.In this study,we inoculated the horse chestnut with soft rot bacteria and used transcriptome analysis to identify MLO genes potentially related to susceptibility to soft rot disease,as well as DXS,WRKY,and NAC genes related to disease resistance.We validated the function of these genes through gene cloning,q RT-PCR analysis,and construction of a silencing system in transformed tobacco seedlings.1.To observe the infection of carrot pectin soft rot bacteria,Pcc strains containing a green fluorescent protein(GFP)reporter and regular Pcc were inoculated onto horse chestnut leaves.The results showed that visible disease symptoms appeared at 12 hours post-inoculation,with water-soaked lesions appearing around the injection site and spreading along the veins at 24 hours,and the leaves becoming wet,rotten,and malodorous at 36 hours.The Pcc strain containing the GFP reporter had weaker infectivity compared to the regular Pcc,but both strains produced consistent disease symptoms after infection.2.Transcriptome sequencing analysis was performed on the control group and calla lily inoculated at 0 h,12 h and 24 h,yielding 31,157 transcriptome sequences,of which 18,945 were annotated.Using sterile water as a control,the differentially expressed genes were 9169,4897,10663 after 12 hours,24 hours,and 36 hours of inoculation,respectively.Upregulated genes are more than downregulated genes.Structural analysis of the sequences revealed that the total number of alternative splicing events in calla lily increased significantly after inoculation with the bacteria,with a total of 41,965 alternative splicing events identified in the 12-hour library compared to the control group.Exon skipping(SE)events accounted for the largest increase in alternative splicing,comprising 52 % of the total events.Expression level analysis of each sample showed that low-abundance expressed genes accounted for 24 %of the average expression level,while genes with medium and high abundance accounted for 18 % and 25 %,respectively.GO enrichment analysis showed that differentially expressed genes were mainly enriched in biological processes such as iron ion binding and carbohydrate metabolic process,as well as molecular functions such as heme binding and oxidoreductase activity.KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in carbon metabolism and plantpathogen interaction.Combined with annotation information and related studies,one gene,named ZaMLO,was identified as potentially associated with calla lily soft rot disease susceptibility,and three genes,named ZaDXS,ZaWRKY,and ZaNAC,were identified as potentially associated with resistance to the disease.3.Four selected genes were analyzed by qRT-PCR.Actin was used as the internal reference gene,and the expression levels of the four genes were measured at 12 h,24 h,and 36 h after inoculation with Pcc.The results showed that the expression levels of ZaDXS and ZaNAC genes continued to increase,while the expression level of ZaWRKY gene initially increased and then decreased,and the expression level of ZaMLO gene initially decreased and then increased.All four genes showed a significant upregulation in expression at each time point after Pcc infection compared to the control group.It is speculated that all four genes may be involved in the process of calla lily soft rot disease resistance/susceptibility.4.Cloning of ZaDXS,ZaWRKY,ZaNAC,and ZaMLO genes resulted in the sequences of 2133 bp,978 bp,1044 bp,and 1791 bp,respectively.Bioinformatics analysis revealed that the proteins encoded by these genes did not contain any signal peptides.The structural domain prediction results indicate that ZaDXS contains typical PLN02582 structural domains and belongs to the PLN02582 superfamily;ZaWRKY contains a DNA binding domain(WRKY),belonging to the WRKY superfamily;ZaMLO contains an MLO domain,belonging to the MLO superfamily;ZaNAC contains a NAM domain and belongs to the NAM superfamily.Promoter element analysis revealed that all four genes had jasmonic acid pathway cis-acting regulatory elements and abscisic acid cis-acting regulatory elements.The results of the protein phylogenetic tree show that the amino acid sequence encoded by ZaDXS has low similarity to other species,and the protein has a relatively late evolutionary time compared to other species;The amino acid sequence of ZaWRKY has the highest similarity to that of lotus;The amino acid sequence of ZaNAC has the highest similarity to Aristolochia;The amino acid sequence of ZaMLO has the highest similarity to taro.5.To further verify the gene function of the ZaDXS and ZaMLO genes,transient silencing validation was performed on the tobacco(N.benthamiana)for Nb DXS and Nb MLO.The results showed that plants with silenced Nb DXS genes were more susceptible to disease than the control group,while plants with silenced Nb MLO genes showed some resistance to soft rot disease.