A Study Of The Pectin Lyase PL1B Gene Of Pseudomonas Discospora As A SIGS Target

Tea leaf spot is a common tea leaf disease,which seriously affects the yield and quality of tea.When the disease occurs seriously,it will cause the shedding of tea leaves,weakening the tea trees.Therefore,it is of great significance to isolate and identify the pathogen of tea leaf spot,screen the effective biogenic pesticides and further study the mechanism of antifungal activity.In this paper,several strains were isolated and purified from tea leaf spot in Erlong tea region,Yuqing County(27.38 °N,107.36 °E,altitude: 860 m),Guizhou Province.The colony morphology of the representative isolate GZYQ2018YQX001 on the PDA was white with feathery edges,the back of the colony was pale yellow coloration.With the extension of incubation time,the aerial hyphae become dense,and the back of the colony turned yellow coloration.At the later stage of incubation,black fruiting body were produced,and the undersurface of the colony displayed brown coloration.Conidiogenous cells were fusiform,with thin walls,and the conidia were fusiform,thin walls and septal.The mature conidia dimensions were 23.56±2.89(range 17.49-30.16)?m × 6.3±0.75(4.74 to 7.4)?m in size.Maximum parsimony phylogenetic analysis based on concatenated sequences of combined translation elongation factor 1-alpha(TEF)(1-995),internal transcribed spacer(ITS)(996-1545),and partial ?-tubulin gene(TUB)(1546-2027).The results showed that the representative strains GZYQ2018YQX001 was identical to reference strains Pestalotiopsis trachicarpicola MFLUCC12-0264,and the clade was supported by 100% bootstrap values.The pathogenic experiment showed that the strain GZYQ2018YQX001 was the pathogen of tea leaf spot.The results showed that the strain GZYQ2018YQX001 infected tea leaves mainly through stomata by morphological observation of different periods by scanning electron microscopy.RNA was extracted from tea leaves infected by pathogenic fungi at different stages,then reverse-transcribed to create the c DNA library.RNA sequencing was performed on Illumina Novaseq^TM 6000 Hiseq platform,and the functions of different expressed genes(DEGs)were analyzed by GO and KEGG database.A large number of DEGs were found to be related to pectate lyase,and the gene of PL1 B was the most significantly expressed.The gene of PL1 B was closely related to pathogen infection process.In this study,genetic transformation and virus-induced gene silencing(VIGS)techniques were used to identify the gene function of P.trachicarpicola.The plant expression vector p CAMBIA2301-KY was constructed,and PL1 B gene was transferred into Nicotiana benthamiana.6 positive plants were identified by PCR.The expression level of PL1 B gene in 6 positive plants was detected by RT-q PCR,the results showed that the relative expression level of PL1 B gene in plant number “2” was the highest,followed by plant number “1”.The phenotype of 6 positive plants were short,with more branches and albinism in leaves compared with wild-type plants.The TRV-VIGS transient expression vector of PL1 B gene was constructed,and the phenotype of PL1 B gene silenced tobacco was observed after 10 days of silencing.The results showed that there was no significant difference in phenotype compared with the control.q PCR showed that the expression levels of the target fragments of PL1B-17-4B-2 and PL1B-17-3B-3 were significantly increased after injection of tobacco,pathogen inoculation did not induce severe disease in the leaves of TRV: PL1B-17-4B-2,TRV: PL1B-17-3B-3 plants,whereas negative control(TRV:0)leaves exhibited necrotic symptoms.and CK treatment was no symptoms.In conclusion,PL1 B gene is a potential target for spray-induced gene silencing(SIGS),and the results of this study also lay a foundation for the development of plant disease resistance genetic improvement based on pathogen specific sequence.