The Molecular Mechanism Of Chitosan Improving The Resistance Of Grapes To Botrytis Cinerea And The Functional Analysis Of VvPGIP

Grape(Vitis vinifera L.)as an important fruit tree is widely cultivated worldwide,and China is one of the largest producers and consumers.As a kind of nonmenopausal fruit,grapes will not continue to mature after receipt,so they must be harvested at a stage close to maturity.In addition to environmental factors,fungal rot is one of the main reasons for the deterioration of grapes.Botrytis cinerea-induced gray mold is the main problem faced by most fruit trees after harvest(Ahmed 2018).It is considered to be the most important post-harvest fungal pathogen,which causes a large loss of fresh fruits,vegetables and ornamental plants.Due to its broad-spectrum antibacterial activity,non-toxicity and harmlessness,chitosan(β-1,4-D-glucosamine)is considered to be a promising material for edible coatings in the fruit postharvest field.Chitosan treatment is believed to significantly improve the disease resistance of post-harvest fruits,but the molecular mechanism of its action is still unclear.At the same time,PGIP is a key gene of plant defense response,which can inhibit the degradation of plant cell walls by pathogens,but its mode of action still needs to be studied in depth.Therefore,this research hopes to provide help for improving the disease resistance of grapes after harvest.1.To evaluate the effect of chitosan treatment on the resistance of two grape varieties(’Kyoho’ and ’Shine Muscat’)to Botrytis cinerea through transcriptomics and metabolomics analysis.In addition,we analyzed the effect of chitosan treatment on the growth of Botrytis cinerea hyphae by scanning electron microscopy,and measured the disease indicators and antioxidant enzyme activities of grape berries after inoculation.The results showed that the chitosan treatment reduced the damage of Botrytis cinerea to grape berries.At the same time,chitosan may destroy the mycelium of Botrytis cinerea and increase the antioxidant enzyme activity of grape fruit.Differentially expressed genes(DEGs)from processed or unprocessed chitosan samples are mainly enriched in disease perception,plant hormone biosynthesis and signal transduction,and secondary metabolism.In addition,we found that chitosan treatment also significantly down-regulated the cell wall degradation genes in ’Shine Muscat’ and maintained fruit firmness.We use different metabolites in "Kyoho" and’Shine Muscat’ to detect a large number of secondary metabolites,such as epigallocatechin gallate,catechins,resveratrol,etc.In short,chitosan treatment improved the disease resistance of grape berries.At the same time,we established a possible model for chitosan to regulate the disease resistance of ’Kyoho’ and ’Shine Muscat’,which helps to clarify the effect of chitosan on gray grapes.Possible molecular mechanism of spore tolerance.2.In order to explore the function of VvPGIP,we used IL-60 system to transiently express VvPGIP in grape and strawberry fruits and used Botrytis cinerea to infect the transiently expressed fruits.At the same time,we determined the expression of genes related to cell wall metabolism and the content of cell wall composition.The results showed that transient expression of VvPGIP can significantly reduce the incidence and disease index of grape and strawberry fruits,and reduce the damage of Botrytis cinerea.At the same time,transient expression of VvPGIP also downregulated the expression of cell wall hydrolysis genes and PG enzyme activity,and inhibited the decrease of total pectin and cellulose content.In addition,we found that Botrytis cinerea infection also up-regulated a variety of plant pectin hydrolase enzymes.The interaction protein prediction results show that VvPGIP interacts with a variety of plant transcription factors and cell wall metabolic proteins that respond to biological stress.In conclusion,VvPGIP significantly inhibits the degradation of plant cell walls by Botrytis cinerea to improve plant disease resistance.3.We screened the interacting proteins of VvPGIP using the yeast two-hybrid system and verified the results using BIFC.The results showed that a total of 28 candidate interacting proteins were obtained by screening the library.We selected 6proteins for point-to-point verification,including 1,3-β-endo-glucanase(Endo-1,3-Beta-Glucanase,Vv GLU)and pathogenesis-related protein 4(Pathogenesis-related protein,VvPR-4)It can grow normally on QDP/X medium and the colony is blue.Finally,we verified the interaction of VvPGIP with Vv GLU and VvPR-4 on the cell membrane using BIFC technology.Vv GLU belongs to PR-2 in the PR protein family and can degrade the callosum layer in the cell wall,while VvPR-4 catalyzes the hydrolysis of chitin in the cell wall of insects and fungi.In short,VvPGIP interacts with a variety of cell wall hydrolyzed proteins to inhibit the degradation of plant cell walls by pathogens,and at the same time degrade chitin in the cell walls of pathogens through VvPR-4.