Molecular Mechanism Of Boron Regulating Watermelon Resistance To Cucumber Green Mottle Mosaic Virus(CGMMV) Infection

The yield and quality of watermelon were seriously affected by cucumber green mottle mosaic virus（CGMMV）infection.Boron has important impacts on plant growth and development,especially fruit formation.Previous studies in our laboratory revealed that boron was closely related to the symptoms of decay and blood flesh in watermelon fruits induced by CGMMV infection,and boron application could significantly reduce the percentage and severity of blood flesh disease,however,the mechanism was unclear.In this study,the differential expression genes（DEGs）in watermelon responding to CGMMV infection and boron regulation against CGMMV were identified by multi-omics and bioinformatics analyses.Virus-induced gene silencing（VIGS）was used to verify the antiviral function of key candidate genes.Combined with high performance liquid chromatography（HPLC）,inductively coupled plasma mass spectrometry（ICP-MS）and biochemical detection,we further explored the molecular mechanism of key candidate genes in resistance/susceptibility to CGMMV infection in watermelon.1.Boron can inhibit CGMMV infection in watermelon,and significantly alleviate mosaic symptoms on leaves and blood flesh symptoms in fruits.To explore the effects of boron application on CGMMV infection in watermelon,watermelon seedlings were cultured with 0 mg·L^-1,0.5 mg·L^-1（normal）and 1.0 mg·L^-1 boron concentration gradient nutrient solution,and watermelon leaves were sprayed with 30 mg·L^-1and 300 mg·L^-1 H₃BO₃ at seedling stage and vine extension stage,respectively.The results showed that the accumulations of CGMMV RNA in watermelon leaves were reduced by 55%and 95%,and the accumulations of CGMMV CP protein were reduced by 30%and 74%in treatments by normal and double concentration of boron,respectively.In watermelon fruits,the accumulations of CGMMV RNA and CP were respectively reduced by 58%and 52%,and the blood flesh rate were reduced by 45%after spraying boron.Therefore,both hydroponic supplement and exogenous application of boron can inhibit CGMMV infection in watermelon.2.Boron plays significant regulatory roles in the homeostasis of nutrients and carbohydrates to inhibit CGMMV infection in watermelon.Boron has important effects on absorption and transport of nutrient elements in watermelon leaves after CGMMV infection.Compared with the treatment of only inoculated with CGMMV,the hydroponic supplement of boron upregulated the expression of element absorption and transport genes,i.e.,aquaporin NIPs（NIP2;1 and NIP5;1）,boron transporter 2（BOR2）,K⁺uptake permease 11（KUP11）and vacuolar iron transporter 1（VIT1）,increased the contents of B,N,K,Ca and Fe,and promoted the normal growth of leaves.In fruits,exogenous application of boron also induced NIP5;1 and BOR2 expression,deduced NRT1.5,KUP11 and VIT1 expression,increased the content of B,decreased the contents of N,K,Ca and Fe,and maintained the homeostasis of nutrients in cells.Boron regulated carbohydrate transformation and transport in watermelon.In leaves and fruits,boron supplement altered the expression of beta-fructofuranosidase,insoluble isoenzyme CWINV1,upregulated the expressions of bidirectional sugar transporter SWEETs,and improved the transformation and transport of soluble sugar compared with the treatment of only inoculated with CGMMV.In addition,boron application also reduced pectin content in leaves;the expression of cellulose synthase（Ces A）and COBRA-like protein 7（COBL7）related to cell wall mechanical strength were upregulated,the expression of polygalacturonase（PG）and pectate lyase-like protein 1（PL1）related to pectin catabolism were downregulated,the contents of lignin,cellulose and hemicellulose were increased,and the polysaccharide content was restored in fruits.3.In watermelon,NIP5;1 and SWEET4 play significant roles in resistance to CGMMV infection through regulating nutrient and carbohydrate homeostasis.In aquaporin NIP5;1-and bidirectional sugar transporter SWEET4-silenced plants,the mosaic symptoms on leaves and blood flesh symptoms in fruits were significantly aggravated,and CGMMV accumulations were strongly higher than that in controls,respectively.Especially in NIP5;1-silenced plants,CGMMV accumulation was 5-to 10-fold of that in controls,and the area of blood flesh symptom reached more than 90%.Silencing NIP5;1 significantly increased the contents of N,P,K,Ca,Mg and Fe,and decreased the contents of S and B in fruits.The alteration patterns of element contents in SWEET4-silenced plants were similar to,while not as significant as,that in NIP5;1-silenced plants.In fruit,silencing NIP5;1 and SWEET4 both decreased soluble sugar content;silencing NIP5;1 caused the decrease of cellulose and hemicellulose content,and silencing SWEET4 caused the decrease of pectin content.The changes of these physiological indexes were consistent with the changes of nutrient elements,soluble sugar and cell wall polysaccharide levels in CGMMV-infected watermelon fruits in the field.4.The results of transcriptome,proteome and metabolome analyses for watermelon fruits under different treatments showed that the DEGs related to the pathogenesis of CGMMV and watermelon resistance to CGMMV infection regulated by boron were mainly involved in the following biological processes:plant hormone synthesis and transmission,immune signal transmission,synthesis and metabolism of cell wall and plasma membrane,and energy metabolism and secondary metabolism.Multi-omics analyses showed that xyloglucan endotransglucosylase/hydrolase protein 23（XTH23）,pectinesterase/pectinesterase inhibitor 7（PE/PEI7）,tubulin beta-1 chain（TUBB1）,ATP sulfurylase 1（ATPS1）,aldo-keto reductase 1（AKR1）,transcription factors MYB6 and WRKY12 and histone H2A（H2A）were associated with the differential accumulation of metabolites.5.Functional verification of key genes regulated by boron and important host factors related to CGMMV infectionThe results of CGMMV-based VIGS assays indicated that ethylene-responsive transcription factor RAP2-3（RAP2-3）,XTH23,PE/PEI7,ATPS1,Chaperone protein dna J 11（Dna J11）,MYB6,WRKY12,and H2A regulated by boron were involved in watermelon resistance to CGMMV infection.CGMMV infection resulted in the upregulation of spermidine synthase（SPDS）,Tu BB1 and subtilisin-like protease（SBT）in watermelon fruits,and promoted the symptoms of decay and blood flesh in watermelon fruits,which were important host factors for the pathogenesis of CGMMV.The functions of above watermelon homologous genes were further verified in Nicotiana benthamiana by tobacco rattle virus（TRV）VIGS assays,and the results of the resistance/susceptibility of these genes to CGMMV were consistent with those in watermelon gene VIGS tests.In conclusion,this study revealed the molecular mechanism of boron regulating watermelon resistance to CGMMV infection,which provided a novel insight on the mechanism of boron to alleviate the symptoms of decay and blood fresh induced by CGMMV infection in watermelon fruits.The candidate genes related to assistance or restriction of CGMMV infection were determined,which provided candidate targets for molecular disease resistance breeding of watermelon and other cucurbit crops,and offered a new and environmentally friendly strategy for green prevention and control of CGMMV.