Function And Mechanisms Of Apoplastic Glucose Signaling In Tomato Disease Resistance Under Low Light Condition

In recent years,the facility vegetable industry is flourishing,and tomato is an important economic vegetable crop in China.However,with global climate change,various abiotic and biotic stresses are occurring more frequently in tomato industry due to the extreme weather,causing huge economic losses.Plant diseases often outbreak in tomato industry under low light conditions caused by facility coverage,rainy weather and frequent haze,especially the leaf speck disease,caused by Pseudomonas syringae pv.tomato(Pst)DC3000,which is harmful and hard to be controlled.Chemical pesticides application remains the common method to control plant diseases,which poses serious threats to food security and ecological environment.Here,our results reveal that tomato plant cells can sense and transduct apoplastic glucose signaling through RGS1(Regulator of G protein signaling)/G protein signaling pathway to regulate the defense against Pst DC3000 under low light condition.And we found that the NAC transcription factor SlNAP1 can regulate the defense against abiotic and biotic stresses.The main results are as follows:1.The disease resistance of tomato plant is reduced under low light condition,however,the mechanism is not clear.We found that apoplastic glucose signaling played an important role in tomato disease resistance under low light condition.Compared with normal light(300?mol m^-2 s^-1),low light(50?mol m^-2 s^-1)significantly reduced the defense against Pst DC3000.The contents of apoplastic glucose and total glucose in tomato leaves were measured after Pst DC3000 inoculation under normal light and low light conditions.Both apoplastic glucose and total glucose levels decreased in low light or after Pst DC3000 inoculation.After Pst DC3000 inoculation,the apoplastic glucose levels decreased more dramatically than total glucose levels,apoplastic glucose levels and total glucose levels decreased 91.7% and 40.9%in low light relative to those in normal light,respectively.In addition,we found that exogenous glucose treatment significantly enhanced the defense against Pst DC3000 under low light.These data indicate that apoplastic glucose may act as a signaling molecule,and plays a critical role in tomato resistance to Pst DC3000 under low light condition.2.AtRGS1 is considered to be an extracellular glucose sensor in Arabidopsis,but the direct evidence for RGS1 binding to glucose is lacking.We found that tomato RGS1can directly bind to glucose at cellular levels.RGS1 negatively regulated disease resistance and was required for glucose-enhanced immunity under low light.We used the amino acid sequence of the Arabidopsis AtRGS1(At3g26090)protein as the query sequence to identify the tomato Sl-RGS1 gene(Solyc05g014160)through Blast method.The subcellular localization of RGS1 was identified by transiently overexpressing RGS1-GFP in Nicotiana benthamiana.We found that tomato RGS1 was localized in plasma membrane.The cell-based biolayer interferometry assay revealed that tomato RGS1 specifically binds to glucose.And laser confocal microscopy imaging showed that glucose induced the endocytosis of RGS1 protein in a concentration-and time-dependent manner.By generating rgs1 gene editing mutant lines,we found that tomato RGS1 negatively regulated disease resistance,and the glucose-enhanced immunity effect was largely compromised in rgs1-4 mutant plants under low light condition.3.Sugar sensing and signaling are not isolated,AtRGS1 generally couples with G protein to mediate sugar signaling.We found that RGS1 coupled with G protein complex sensed and transduced apoplastic glucose signaling to regulate the disease resistance of tomato plants under low light condition.We identified the tomato G protein family and found that the tomato genome encodes six G?subunit gene(one canonical G?GPA1 and five Extra-Large G protein subunit genes),one G?subunit gene AGB1,and four G?subunit genes.Through Bi FC and split-luc methods,we found that RGS1 interacted with GPA1,and GPA1 interacted with AGB1,but the interaction between RGS1 and AGB1 was not detected.Exogenous glucose treatment can weaken the interaction strength of RGS1-GPA1 as well as GPA1-AGB1.By generating gpa1and agb1 mutant lines,we found that tomato GPA1 negatively regulated the resistance to Pst DC3000 under low light,while tomato AGB1 acted as a positive regulator of the disease resistance.Glucose can significantly enhanced the resistance of tomato wild-type materials to Pst DC3000 under low light condition.However,the glucose-enhanced defense effect was severely impaired in the above-mentioned mutant lines.Our results show that tomato GPA1 and AGB1 may act downstream of RGS1 to transduct apoplastic glucose signaling and regulate the resistance to Pst DC3000 under low light condition.4.Transcription factors located in the nucleus can mediate the function of sugar signaling by regulating the expression of downstream target genes.We identified a NAC transcription factor SlNAP1 whose expression was significantly down-regulated in tomato G protein?subunit agb1 mutant materials,and we found that SlNAP1 can regulated the defense against abiotic and biotic stresses.We found that tomato SlNAP1was localized in plasma membrane and nucleus by transiently overexpressing SlNAP1-GFP in Nicotiana benthamiana.By generating SlNAP1 transgenic lines,we found that the SlNAP1-overexpressing plants were shorter than the wild-type plants.SlNAP1-overexpressing plants showed significantly enhanced defense against leaf speck disease and root-borne bacterial wilt disease.In addition,SlNAP1 overexpression dramatically improved drought tolerance in tomato.Analysis of different hormone contents revealed a reduced level of physiologically active gibberellins(GA₄)and an increased level of salicylic acid(SA)and abscisic acid(ABA)in the SlNAP1-overexpressing plants.Moreover,EMSAs and Ch IP-q PCR assays showed that SlNAP1 directly targeted SlGA2ox3,SlPAL3 and SlNCED1.Our results show that SlNAP1 may promote GA4deactivation,SA and ABA biosynthesis to regulate plant growth and the defense against abiotic and biotic stresses in tomato.In summary,our results revealed that tomato plant cells can sense and transductapoplastic glucose signaling through RGS1/G protein signaling pathway to regulate plant immunity under low light condition.And we identified a vital tomato NAC transcription factor,SlNAP1,positively regulating the defense against leaf speck disease,root-borne bacterial wilt disease,and drought stress.This research investigates how plants stimulate their immunity responses to conquer abiotic and biotic stresses.And we hope that this research can help us develop green and safe ways to enhance broad-spectrum resistance of crop in agriculture.