Identification and functional analysis of protein secreted by Alternaria solani

Abstract

Early blight, caused by the necrotrophic fungus Alternaria solani, is an important foliar disease that causes major yield losses of potato. Effector proteins secreted by pathogens to host cells can inhibit host immune response to pathogens. Currently, the function of effector proteins secreted by A. solani during infection is poorly understood. In this study, we identified and characterized a novel candidate effector protein, AsCEP50. AsCEP50 is a secreted protein that is highly expressed throughout the infection stages of A. solani. Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana and tomato demonstrated that AsCEP50 is located on the plasma membrane of N. benthamiana and regulates senescence-related genes, resulting in the chlorosis of N. benthamiana and tomato leaves. Δ50 mutants were unaffected in vegetative growth, spore formation and mycelium morphology. However, the deletion of AsCEP50 significantly reduced virulence, melanin production and penetration of A. solani. These results strongly supported that AsCEP50 is an important pathogenic factor at the infection stage and contributes to the virulence of Alternaria solani.

Fig 1. Expression patterns of the AsCEP50 gene during the infection of potato leaves.

The expression level at the hyphal stage was used as a control to analyze the specific expression at the infection stage. Student’s t-test was used as a significance test of difference (n=6). **, indicates a significant difference (0.001

Fig 2. Subcellular localization of AsCEP50 proteins in N. benthamiana.

The Agrobacterium strain EHA105 containing the pCAMBIA1301 vector as the control and the AsCEP50 gene were transiently expressed in the leaves of N. benthamiana. Bar=20 μm.

Fig 3. Agrobacterium containing AsCEP50 effector promotes senescence of N. bethamiana and tomato leaves.

(A) Nicotiana benthamiana and tomato leaves were infiltrated by control (EV; left, leaf tip to petiole direction) and pCAMBIA1301 Agrobacterium containing AsCEP50-NSP gene (right, leaf tip to petiole direction). (B) Relative mRNA levels of SEN4, SAG12 and DHAR1. Expression levels of senescence- and oxidative stress-associated genes were examined in N. benthamiana (EV and AsCEP50-NSP) 4 days after injection with Agrobacterium.

Fig 4. AsCEP50 gene replacement and PCR screening of mutants.

(A) The diagram of homologous recombination of AsCEP50 and Hyg gene. (B) Verification of Hyg gene in the genomes of wild-type and Δ50 mutant strains.

Fig 5. Determination of the phenotype and melanin of the wild-type, Δ50 mutant and revertant strains.

(A) the solution of wild-type, Δ50 mutant and revertant strains that determine the melanin content. (B) the absorbance of the wild-type, Δ50 mutant and revertant strain solutions at 400 nm.

Fig 6. Penetration of the WT, Δ50 mutant and revertant strains.

The colony morphology of WT (left), Δ50 mutant (middle) and revertant strains (right) filtered through three layers of sterile cellophane.

Fig 7. Pathogenicity detection of AsCEP50 gene.

(A) The detached potato leaves were inoculated with the spore suspensions of Δ50 mutant strains (left, leaf tip to petiole direction), wild-type strains (upper right, leaf tip to petiole direction) and revertant strains (lower right, leaf tip to petiole direction). (B) The lesion diameters of potato leaves infected by the wild-type, Δ50-1 mutant, Δ50-2 mutant, revertant-1 and revertant-2 strains. ***, indicates an extremely significant difference (p<0.001) compared with the control (HWC-168). Bar means standard deviation (SD).