Identification of effector CEP112 that promotes the infection of necrotrophic Alternaria solani

Abstract

Background: Alternaria solani is a typical necrotrophic pathogen that can cause severe early blight on Solanaceae crops and cause ring disease on plant leaves. Phytopathogens produce secretory effectors that regulate the host immune response and promote pathogenic infection. Effector proteins, as specialized secretions of host-infecting pathogens, play important roles in disrupting host defense systems. At present, the role of the effector secreted by A. solani during infection remains unclear. We report the identification and characterization of AsCEP112, an effector required for A. solani virulence.

Result: The AsCEP112 gene was screened from the transcriptome and genome of A. solani on the basis of typical effector signatures. Fluorescence quantification and transient expression analysis showed that the expression level of AsCEP112 continued to increase during infection. The protein localized to the cell membrane of Nicotiana benthamiana and regulated senescence-related genes, resulting in the chlorosis of N. benthamiana and tomato leaves. Moreover, comparative analysis of AsCEP112 mutant obtained by homologous recombination with wild-type and revertant strains indicated that AsCEP112 gene played an active role in regulating melanin formation and penetration in the pathogen. Deletion of AsCEP112 also reduced the pathogenicity of HWC-168.

Conclusion: Our findings demonstrate that AsCEP112 was an important effector protein that targeted host cell membranes. AsCEP112 regulateed host senescence-related genes to control host leaf senescence and chlorosis, and contribute to pathogen virulence.

Keywords: Alternaria solani; Chlorosis; Effector; Gene knockout; Pathogenicity; Senescence.

Fig. 1

Expression patterns of the AsCEP112 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. Actin expression was used as an internal reference for normalizing within the samples. Student’s t-test was used as a significance test of differences (n = 6). *, indicates a difference (0.01 < p < 0.05) compared with the control (hyphal stage); **, indicates a significant difference (0.001 < p < 0.01) compared with the control; ***, indicates an extremely significant difference (p < 0.001) compared with the control. Bars represent standard deviations (SDs)

Fig. 2

Subcellular localization of AsCEP112 proteins in N. benthamiana. The Agrobacterium strain EHA105 containing the pCAMBIA1301 vector, as the control, and the AsCEP112 gene were independently transiently expressed in N. benthamiana leaves. Bar = 20 μm

Fig. 3

Agrobacterium containing AsCEP112 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 AsCEP112-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 AsCEP112-NSP) and coi1 shoots 4 days after injection with Agrobacterium

Fig. 4

AsCEP112 gene replacement and RT-PCR screening of mutants. A Diagram of the homologous recombination of AsCEP112 and Hyg genes. B Verification of the Hyg gene in the genomes of WT and AsCEP112 mutant strains

Fig. 5

Determination of the phenotypes and melanin levels of the wild-type, AsCEP112 mutant and revertant strains. A The solutions of wild-type, AsCEP112 mutant and revertant strains for the determination of the melanin contents. B The absorbance values of the wild-type, AsCEP112 mutant and revertant strain solutions at 400 nm. *, indicates a difference (p < 0.05) compared with the control (HWC-168); ***, indicates an extremely significant difference (p < 0.001) compared with the control (HWC-168). Bars represent standard deviations (SDs)

Fig. 6

Penetration of the WT, AsCEP112 mutant and revertant strains. The colony morphology of wild-type, AsCEP112 mutant and revertant strains filtered through three layers of sterile cellophane

Fig. 7

Detecting the pathogenicity of the AsCEP112 gene. A The isolated potato leaves were inoculated with the spore suspensions of AsCEP112 mutant (left, leaf tip to petiole direction), wild-type (upper right, leaf tip to petiole direction) and revertant (lower right, leaf tip to petiole direction) strains. B The diameters of lesions on potato leaves infected by the wild-type, mutant Δ112-2, mutant Δ112-3, revertant-2 and revertant-3 strains. ***, indicates an extremely significant difference (p < 0.001) compared with the control (HWC-168). Bars represent standard deviations (SDs)