The Plant Resistance Against Verticillium Wilt Is Improved By Using Cotton Kiwellin Protein And Fungal P4-ATPase

Cotton is one of the most important economic crops in the world.Cotton production occupies a very important position in the national economy of China.V.dahliae is a soil-borne pathogenic fungus.Due to its broad host range,frequent mutation,complex pathogenic mechanism,and the lack of germplasm resources with high resistance to Verticillium wilt in upland cotton,Verticillium wilt causes great losses of cotton production in China.Therefore,Verticillium wilt is known as the"cancer"of cotton.Previous studies have shown that maize Kiwellins(KWLs)are involved in the interaction between host and pathogen,and overexpression of the genes can improve the resistance of maize to smut disease.However,it is not clear whether there are homologous genes of KWLs in cotton?what the function in the interaction between cotton and V.dahliae do KWLs have?Our previous study showed that BbCrpa,a P4-ATPase of the insecticidal fungus B.bassiana,is able to deliver anti-fungal agent Cs A and FK506 to the vacuole via vesicle-mediated transport pathway for cell detoxification.Mycotoxins produced by V.dahliae are considered as an important pathogenic factor of V.dahliae.Can the expression of the fungal P4-ATPase genes in cotton enhance plant resistance to Verticillium wilt by means of cell detoxification?This study is aimed to answer these questions.The main results are as follows:1.The expression of GhKWL1 is induced by V.dahliae,and GhKWL1 is located in the nucleus.The KWL protein sequences of maize and kiwifruit were used as the template for the homology alignment in the genomic database of upland cotton.A total of 35 KWL genes were identified.Of them,the expression levels of GhKWL1,GhKWL2,GhKWL3,and GhKWL4 were remarkably upregulated after inoculation with V.dahliae.The subcellular localization results showed that only Ghk WL1 showed localization in the nucleus,suggesting that Ghk WL1 might participate in the regulating the expression of other genes.Based on the above results,GhKWL1 was selected for further study.2.Downregulation of GhKWL1 decreases the resistance of cotton to Verticillium wilt,while overexpression of GhKWL1 increases the resistance of Arabidopsis to the disease.Silenced the transcription of GhKWL1 in upland cotton by VIGS,the cotton plants were inoculated with V.dahliae.Compared with the control plants,the disease index of GhKWL1-silenced plants was significantly increased.The disease symptoms of the GhKWL1-silenced cotton,such as leaf yellowing,wilting and vascular browning,were more severe,and the biomass of V.dahliae in the leaves was also significantly increased.On the other hand,overexpression of GhKWL1 in Arabidopsis led to a significant decrease in the wilting rate.The severity of the disease symptoms was much lower than the wild-type control,and the biomass of V.dahliae in the leaves was also significantly decreased than the control.These data suggest that GhKWL1 has a positive effect on the plant resistance to Verticillium wilt.3.GhKWL1 can upregulate the expression of GhERF105,and repression the expression of GhKWL1 increases cotton susceptibility to Verticillium wilt.The transcriptome data analysis showed that GhKWL1 positively regulates the expression of genes involved in disease-resistance signaling pathways,such as plant-pathogen interaction pathway,plant MAPK signaling pathway,and plant hormone signaling pathway.GO enrichment analysis indicated that the down-regulated genes in GhKWL1-silenced plants were mainly related to transcription factors,including 22 ERF genes.Among them,six genes with greatest down-regulated expression level were verified by q RT-PCR.GhERF105 with the highest down-regulated expression level was selected for dual-luciferase assay.The results showed that the promoter of GhERF105was activated by the expression of GhKWL1 in tobacco.Ch IP-q PCR assay also proved that GhKWL1 could bind to the promoter of GhERF105.These results indicated that GhKWL1 upregulates the expression of GhERF105,while GhERF105 is a positive regulator for the resistance to Verticillium wilt.4.GhKWL1 and GhERF105 both can promote the expression of PR4 and PDF1.2.The expression levels of GhPR4 and GhPDF1.2 in GhKWL1-silenced and GhERF105-silenced cotton plants were down-regulated compared with control.The expression levels of GhERF105,GhPR4 and GhPDF1.2 were all up-regulated in GhKWL1 transgenic cotton.The expression levels of At PR4 and At PDF1.2 in two GhKWL1 transgenic Arabidopsis lines were up-regulated compared with wild type.These results suggest that GhERF105 can positively regulate the expression of PR4 and PDF1.2.Moreover,GhKWL1 and GhERF105 may be located in the same disease resistance signaling pathway.5.VdISC1 physically interactes with GhKWL1 to attenuate GhKWL1 mediated plant defense response.A.tumefaciens carrying VdISC1:n YFP and GhKWL1:c YFP vectors were injected into N.benthamiana.72 h later,yellow fluorescence signal was observed in leaf epidermal cells,suggesting a possible interaction between VdISC1 and GhKWL1 in tobacco.Total proteins were extracted from tobacco leaves holding VdISC1:FLAG and GhKWL1:GFP expressing vectors.Co IP experiment showed that VdISC1 could interact with GhKWL1.In addition,the fusion proteins GST:Ghk WL1 and His:VdISC1 were expressed by prokaryotic expression system,and the pull down experiment results showed that VdISC1 and GhKWL1 could bind directly in vitro.Meanwhile,Bi FC and Co IP assays showed that the mutant VdISC1^3A which lost its catalytic activity could still interact with GhKWL1.Dual-luciferase assay showed that both VdISC1 and VdISC1^3Acould inhibit the activation of GhERF105 promoter activated by GhKWL1.These results reveal that VdISC1 inhibits GhKWL1 mediated disease resistance pathway by binding with GhKWL1,and this inhibition is independent of the catalytic activity of VdISC1.6.BbCrpa and VdCrpa can promote the resistance to V.dahliae infection through transporting the V.dahliae toxins into the vacuole.Our previous studies have shown that BbCrpa,a P4-ATPase in Beauveria bassiana,is capable of detoxifying the immunosuppressant Cyclosporin A(Cs A)and FK506 via vesicle mediated transport pathway to transport CSA and FK506 into vacuoles for compartmentation or degradation.Overexpression of BbCrpa can increase the tolerance of V.dahliae to Cs A.VdCrpa,a homologous gene of BdCrpa in V.dahliae,was cloned.It was found that VdCrpa could restore the tolerance of?BdCrpa knock out mutant to Cs A in B.bassiana,suggesting that VdCrpa has a similar detoxification function as BbCrpa.Then,BdCrpa and VdCrpa were overexpressed in Arabidopsis and cotton.The results showed that the tolerance of transgenic Arabidopsis and cotton to V.dahliae toxin was significantly increased.Cinnamate acetate(CIA)is a low molecular weight lipophilic mycotoxin produced by V.dahliae.Observing the distribution of the fluorescently labeled CIA(FITC-CIA)in Arabidopsis cells,it was found that the accumulation of FITC-CIA in the vacuoles of p35S:BbCrpa and p35S:VdCrpa transgenic Arabidopsis was significantly increased.The results showed that both BbCrpa and VdCrpa are capable of promoting the transport of the mycotoxin into vaculoe.7.Overexpression of BbCrpa and VdCrpa enhance the resistance of Arabidopsis,tobacco,tomato,and cotton to Verticillium wilt.BbCrpa and VdCrpa were overexpressed in Arabidopsis,tomato,tobacco and upland cotton,respectively,and the resistance of transgenic lines with high expression levels to Verticillium wilt was analyzed.The results showed that,compared with control plants,the disease symptoms of p35S:BbCrpa and p35S:VdCrpa transgenic lines,such as leaf yellowing and wilting,were mitigatory.The degree of vascular bundle browning in stems of p35S:BbCrpa and p35S:VdCrpa transgenic cotton and tobacco was alleviated.The disease index of p35S:BbCrpa and p35S:VdCrpa transgenic lines was significantly decreased.The biomass of V.dahliae in p35S:BbCrpa and p35S:VdCrpa transgenic cotton leaves was significantly reduced.These data indicated that overexpression of BbCrpa and VdCrpa can increase the resistance of Arabidopsis,tomato,tobacco and cotton to Verticillium wilt.8.Expression levels of defense signaling pathway genes in p35S:BbCrpa and p35S:VdCrpa transgenic cotton were upregulated under V.dahliae infection.After inoculation with the V.dahliae,the transcriptional levels of GhPR1 gene involved in salicylic acid signaling pathway,GhNPR1 gene involved in multiple immune signaling pathway,GhNOA1 gene involved in nitric oxide signaling pathway,and GhAOX1 gene involved in reactive oxygen species signaling pathway in p35S:BbCrpa and p35S:VdCrpa transgenic cotton were up-regulated more than that of wild type.These results suggest that by the promotion of transport of toxins into vacuoles,the expression of BbCrpa and VdCrpa can protect the integrity of plant cell immune system from the damage of toxins to cells,and thus increase the plants resistance to Verticillium wilt.In conclusion,this study reveals that GhKWL1 is a positive regulator of GhERF105-associated pathway;the pathogenic effector protein VdISC1 produced by V.dahliae can disturb GhKWL1 mediated defense response through binding with it;therefore,the enhancement of GhKWL1 expression can protect the host plants from the inhibition,thus increase the resistance to the disease.The study also shows that fungal P4-ATPases,BbCrpa and VdCrpa,can promote the accumulation of V.dahliae toxins in vacuoles,and enhance the plants resistance to Verticillium wilt by protecting the plants from toxicity of Verticillium wilt toxins.This study enriched our understanding of the mechanism of interaction between cotton and V.dahliae,and provided a new strategy for improving the resistance of plants to Verticillium wilt.