Fig. 7
Functional analyses of conserved asparagine residues in the CFEM domain-containing proteins VdSCP76 and VdSCP77 from Verticillium dahliae. A Sequence alignment of the CFEM domain from CFEM-containing VdSCPs in V. dahliae and heme-iron acquisition related CFEM proteins in Candida albicans. The sequences were aligned using Clustal W2 [48]. Black and red asterisks represent the position of the eight conserved cysteine residues and the mutation of the conserved site. B Cell death suppression activity of native and D>N site-directed mutant proteins of VdSCP76 and VdSCP77 were detected by co-expressing transiently with VdEG1 in 4-week-old Nicotiana benthamiana leaves. GFP and VdEG1 were used as controls. C Heat map of resistance-related genes expression in N. benthamiana leaves infiltrated with wild-type and asparagine site-mutated proteins of VdSCP76 and VdSCP77 with co-expression of the cell death inducer VdEG1. Gene expression was detected by reverse transcription-quantitative PCR (RT-qPCR) at 2 days after agro-infiltration. D Virulence assays of wild-type Vd991, VdSCP76, and VdSCP77 gene deletion mutant strains, native VdSCP76 and VdSCP77 complement and asparagine site-mutated complement transformants of V. dahliae on cotton (cv. Junmian 1). Three-week-old seedlings of cotton plants were inoculated, and Verticillium wilt symptoms were photographed at 3 weeks post-inoculation (wpi). E Molecular structural formulas of asparagine and aspartic acid. F CFEM-containing VdSCPs (Asn-type and Asp-type) were expressed transiently in 4-week-old N. benthamiana and the leaves were collected at 2 days after agro-infiltration. The iron transport-associated gene (NbIRT1: iron-regulated transporter 1; NbNramp2: natural resistance-associated macrophage protein 2) expression levels were detected by RT-qPCR. GFP was served as control. G Expression of genes related to the immune response of N. benthamiana was determined by RT-qPCR. The CFEM-containing VdSCPs were co-expressed with VdEG1 transiently, and single infiltration of VdEG1 was used as control. Error bars represent standard errors. * and ** represent significant differences at P < 0.05 and P < 0.01, respectively, between the different VdSCPs treatments and GFP control according to one-way analysis of variance (ANOVA)