Zhihui Yang

Potato early blight is a fungal disease caused by Alternaria solani that seriously affects potato production. Catalase (CAT), an important antioxidant enzyme in plants, regulates the basal immune response to necrotrophic pathogens by decomposing hydrogen peroxide (H₂O₂); however, its role in potato early blight remains unclear. The results revealed that using a H₂O₂ biosensor for transient overexpression in Nicotiana benthamiana, we found that A. solani infection significantly induced sustained H₂O₂ accumulation in tobacco leaves. Exogenous application of H₂O₂ to potato leaves followed by inoculation with A. solani showed that elevated H₂O₂ levels promoted infection by the pathogen. StCAT1 expression was upregulated upon A. solani infection. Silencing StCAT1 in potato reduced antioxidant enzyme activities (SOD, PAL, CAT) and disease resistance, accompanied by elevated H₂O₂ accumulation. Overexpression of StCAT1 enhanced antioxidant enzyme activities and disease resistance while decreasing H₂O₂ levels. Additionally, StABI5 was identified as an upstream transcription factor that activates StCAT1 expression, and silencing StABI5 compromised potato resistance to early blight. In conclusion, this study confirms that StCAT1 acts as a positive regulatory factor and mediates potato resistance to early blight through the StABI5-StCAT1 signaling pathway, providing scientific basis and genetic resources for disease-resistant breeding.

Alternaria solani is an important necrotrophic pathogen causing potato early blight. However, the pathogenic molecular mechanisms of A. solani remain unclear. Previous work identified a specific effector AsCEP20 in A. solani through multi‐omics analysis. AsCEP20 is required for the full virulence of A. solani and targets the host chloroplasts. In this study, we screened out 46 candidate proteins that potentially interact with AsCEP20 in Nicotiana benthamiana using co‐immunoprecipitation followed by liquid chromatography–tandem mass spectrometry analysis. We identified a candidate target protein in potato, filamentation temperature‐sensitive H4 (StFtsH4), which is located in chloroplasts, based on homologous alignment and subcellular localisation analysis. The interaction between AsCEP20 and StFtsH4 was further confirmed by co‐immunoprecipitation, yeast two‐hybrid assay and bimolecular fluorescence complementation assays. The interaction site between AsCEP20 and StFtsH4 is also the chloroplast. Silencing the potato StFtsH4 gene resulted in suppressed pathogen‐associated molecular pattern‐triggered reactive oxygen species (ROS) bursts, and defence‐related genes were significantly downregulated. These results suggest that StFtsH4 positively regulates plant immunity. Therefore, AsCEP20 targets the chloroplast protein StFtsH4 to promote pathogen infection. AsCEP20 attenuates the efficiency of light energy utilisation in photosynthesis by targeting StFtsH4. These results suggest that AsCEP20 suppresses StFtsH4‐mediated potato disease resistance to A. solani . With the increase of light intensity, ROS continued to accumulate in the chloroplast of StFtsH4 ‐silenced plant leaves, while defence‐related genes significantly decreased. Our findings reveal that the impaired StFtsH4 function limits plant photosynthesis, thereby affecting immune signalling.

HhH-GPD (helix–hairpin–helix-glycine/proline/aspartate) family proteins are involved in DNA damage repair. Currently, mechanism of alkylated DNA repair in Crenarchaea has not been fully clarified. The hyperthermophilic model crenarchaeon Saccharolobus islandicus REY15A possesses a novel HhH-GPD family protein (Sis-HhH-GPD), where its Ser152 corresponds to a conserved catalytic Asp in other HhH-GPD homologs. Herein, we report that Sis-HhH-GPD is a novel bi-functional glycosylase, capable of removing both 1-methyladenine (1-meA) from DNA and alkylated bases from DNA created by methyl methanesulfonate (MMS). Mutational analyses show that E134 is essential for catalysis, whereas S152 is not essential. Sis-HhH-GPD might utilize aromatic rings of Y154 and W57 to stack against 1-meA base for flipping-out and then be removed by E134. Additionally, R157, R161 and R200 participate in catalysis. Among four cysteine residues that potentially coordinate with the Fe-S cluster loop, C203, C210 and C219 are involved in catalysis. Importantly, Sis-HhH-GPD is responsible for repair of alkylated DNA created by MMS in vivo. Interestingly, genetic complementary data have confirmed physiological function of Sis-HhH-GPD in alkylated DNA repair and clarified functional roles of its four cysteine residues in vivo. Overall, we provide first evidence that HhH-GPD family protein from Crenarchaea functions in alkylated DNA repair.

Previous work identified a pair of specific effectors AsCEP19 and AsCEP20 in Alternaria solani as contributors to the virulence of A. solani . Here, we constructed AsCEP19 and AsCEP20 deletion mutants in A. solani strain HWC168 to further reveal the effects of these genes on the biology and pathogenicity of A. solani . Deletion of AsCEP19 and AsCEP20 did not affect vegetative growth but did affect conidial maturation, with an increase in the percentage of abnormal conidia produced. Furthermore, we determined the expression patterns of genes involved in the conidiogenesis pathway and found that the regulatory gene abaA was significantly upregulated and chsA , a positive regulator for conidiation, was significantly downregulated in the mutant strains compared to the wild-type strain. These results suggest that AsCEP19 and AsCEP20 indirectly affect the conidial development and maturation of A. solani . Pathogenicity assays revealed significantly impaired virulence of Δ AsCEP19 , Δ AsCEP20, and Δ AsCEP19 + AsCEP20 mutants on potato and tomato plants. Moreover, we performed localization assays with green fluorescent protein-tagged proteins in chili pepper leaves. We found that AsCEP19 can specifically localize to the chloroplasts of chili pepper epidermal cells, while AsCEP20 can localize to both chloroplasts and the plasma membrane. Weighted gene co-expression network analysis revealed enrichment of genes of this module in the photosynthesis pathway, with many hub genes associated with chloroplast structure and photosynthesis. These results suggest that chloroplasts are the targets for AsCEP19 and AsCEP20. IMPORTANCE: Alternaria solani is an important necrotrophic pathogen causing potato early blight. Previous studies have provide preliminary evidence that specific effectors AsCEP19 and AsCEP20 contribute to virulence, but their respective functions, localization, and pathogenic mechanisms during the infection process of A. solani remain unclear. Here, we have systematically studied the specific effectors AsCEP19 and AsCEP20 for the first time, which are essential for conidial maturation. The deletion of AsCEP19 and AsCEP20 can significantly impair fungal pathogenicity. Additionally, we preliminarily revealed that AsCEP19 and AsCEP20 target the chloroplasts of host cells. Our findings further enhance our understanding of the molecular mechanisms underlying the virulence of necrotrophic pathogens.

Bacillus velezensis HN-Q-8, isolated in our previous study, has an antagonistic effect on Alternaria solani. After being pretreated with a fermentation liquid with HN-Q-8 bacterial cell suspensions, the potato leaves inoculated with A. solani displayed smaller lesion areas and less yellowing than the controls. Interestingly, the activity levels of superoxide dismutase, peroxidase, and catalase in potato seedlings were enhanced by the addition of the fermentation liquid with bacterial cells. Additionally, the overexpression of key genes related to induced resistance in the Jasmonate/Ethylene pathway was activated by the addition of the fermentation liquid, suggesting that the HN-Q-8 strain induced resistance to potato early blight. In addition, our laboratory and field experiments showed that the HN-Q-8 strain can promote potato seedling growth and significantly increase tuber yield. The root activity and chlorophyll content of potato seedlings were significantly increased along with the levels of indole acetic acid, gibberellic acid 3, and abscisic acid upon addition of the HN-Q-8 strain. The fermentation liquid with bacterial cells was more efficient in inducing disease resistance and promoting growth than bacterial cell suspensions alone or the fermentation liquid without bacterial cells. Thus, the B. velezensis HN-Q-8 strain is an effective bacterial biocontrol agent, augmenting the options available for potato cultivation.

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.

Blackleg and aerial stem rot of potato (Solanum tuberosum L.), caused by soft rot enterobacteria of the genera Pectobacterium and Dickeya, has recently increased years in Hebei Province, China. Field surveys were performed during the 2021 potato growing season in Hebei to identify and characterize bacterial pathogens. Sixteen potato plants showing blackleg or aerial stem rot were collected from three potato-producing areas, and ten representative pectinolytic bacteria were isolated from symptomatic plants. 16S rDNA sequencing and multilocus sequence analysis were performed to determine the taxonomic position of the bacterial isolates. The isolates belonged to the genus Pectobacterium, including Pectobacterium atrosepticum, Pectobacterium carotovorum, Pectobacterium brasiliense, and Pectobacterium parmentieri. The exceptions were isolates BY21311 and BY21312, which belonged to a new species of Pectobacterium polonicum previously found in groundwater. The taxonomy of isolate BY21311 was confirmed using whole genome-based analysis. P. polonicum has only been identified in potato plants on one farm in Baoding region in China. Isolates BY21311 and BY21312 displayed similar physiological and biochemical traits to the type strain DPMP315^T. Artificial inoculation assays revealed that isolate BY21311 fulfilled Koch’s postulates for potato blackleg. These findings represent the first time P. polonicum, a water-associated Pectobacterium species may be the cause of blackleg in the field. Interestingly, P. polonicum BY21311 has reduced ability to macerate potato tubers when compared to P. atrosepticum, P. brasiliense, P. versatile, and P. parvum, which is more virulent in tubers than the type strain DPMP315^T. The host range of isolate BY21311 was determined by injection method, which can impregnate five plants. Although the genome of isolate BY21311 harbors gene clusters encoding a type III secretion system, it did not elicit a hypersensitive response (HR) in Nicotiana benthamiana or N. tabacum leaves. T3SS effector AvrE and T4SS effector PilN were obtained by predicting isolate BY21311 genome. P. polonicum appears to show significant variations in gene content between two genomes, and gene content varies between isolates BY21311 and DPMP315^T, with strain specific-genes involved in many aspects, including lipopolysaccharide biosynthesis, substrate translocation, T4SS and T6SS among others, suggesting that isolates BY21311 and DPMP315^T might represent distinct clades within the species.

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.