Studies on Functions of Effector Gene ChEP085 in Colletotrichum higginsianum

doi:10.7668/hbnxb.20192657

Abstract

Abstract:

Colletotrichum higginsianum is a hemibiotrophic fungus that causes cruciferous anthracnose, a serious damage to the flowering Chinese cabbage industry in South China. Effectors secreted by pathogens can help pathogens invade host plants and play an important role in the interaction between pathogens and plants. To investigate the role of C. higginsianum effectors in the pathogenesis of cruciferous anthracnose. Based on previous research results from the Laboratory of Tropical and Subtropical Fungi, South China Agricultural University, a potential effector ChEP085 with a 21 amino acid signal peptide at the N-terminus was screened out. To clarify the function of this effector, the cDNA of Arabidopsis thaliana Col-0 infected by C. higginsianum was used as the template, and the expression of ChEP085 gene was determined by qRT-PCR. It was found that the gene ChEP085 had the maximum expression after 24 h of infection. The gene ChEP085 was verified by Agrobacterium tumefaciens mediated Potato virus X(PVX) tobacco transient expression system. The results showed that the gene could stably induce tobacco cell necrosis. The gene ChEP085 was constructed into the enhanced green fluorescent protein vector pBinceGFP and transiently expressed on tobacco leaves for subcellular localization. It was found that the gene was localized in cytoplasm and cell membrane. After deletion of the signal peptide located at the N terminus, the transiently expressed effector ChEP085 was distributed in the nucleus, cell membrane and cytoplasm, indicating that the signal peptide affects the localization of ChEP085. Finally, the gene ChEP085 was knockout by homologous recombination technology, and it was found that the deletion of this gene had no significant effects on the colony morphology and mycelial growth rate of C. higginsianum, but it would lead to the reduction of conidiation and significantly weakened the pathogenicity of C. higginsianum, indicating that ChEP085 play an important role in the conidiation and pathogenesis of C. higginsianum.

Key words: Colletotrichum higginsianum, Effector, ChEP085 gene, Pathogenicity

ZHU Yiming, LIU Yanxiao, HE Jiuqing, DUAN Lingtao, ZHOU Erxun. Studies on Functions of Effector Gene ChEP085 in Colletotrichum higginsianum[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 192-200. doi: 10.7668/hbnxb.20192657.

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Figures/Tables 13

Tab.1 Primers used in this study

引物名称 Primer code	引物序列(5'—3') Primer sequence (5'—3')
Actin-F	CCCCAAGTCCAACAGAGAGA
Actin-R	CATCAGGTAGTCGGTCAAGTCA
qChEP085-F	TCCTGCCTCCCATCGAGTT
qChEP085-R	GTAGTGGTGCTGGTGGTTGT
ChEP085-PVX-F	CTAGCATCGATTCCCGGGCGTCGGTTTCCACTAT
ChEP085-PVX-R	CTCTAGAGGATCCCCGAGAGTAGTGGTGCTGGTGGTTGTG
ChEP085-noSP-PVX-F	CTAGCATCGATTCCCATGGCCGACAACTGCCTCAGAAACG
ChEP085-noSP-PVX-F	CTCTAGAGGATCCCCGAGAGTAGTGGTGCTGGTGGTTGTG
ChEP085-pBincGFP-F	ATAGCCGGTACCCCCATGCGCGCCTCCGTCCTCATC
ChEP085-pBincGFP-R	CTTGCTCACCATCCCGAGAGTAGTGGTGCTGGTGGTTGTG
ChEP085-noSP-pBincGFP-F	ATAGCCGGTACCCCCATGGCCGACAACTGCCTCAGAAACG
ChEP085-noSP-pBincGFP-R	CTTGCTCACCATCCCGAGAGTAGTGGTGCTGGTGGTTGTG
pBincGFP-F	CGAATCTCAAGCAATCAAGCA
pBincGFP-R	GAACCCTAATTCCCTTATCTG
ChEP85us-F	GGCCAGTGCCAAGCTTGACGTCTAAGACCTGGCCTACA
ChEP85us-R	ACCGGAACCAGTCGAGGTTAAGGCTTTGTTGTGTTGATTG
ChEP85ds-F	TATACTCGACTCTAGGGGGTTTGAAAACTCTTTCCTCGG
ChEP85ds-R	GGTACCCGGGGGATCCTTCCAGGAGTATTCTTTGCAGGGT
Hyg-F	GGGCGTCGGTTTCCACTAT
Hyg-R	GATATGTCCTGCGGGTAAATAGC
85-F	ATGCGCGCCTCCGTCCTCATC
85-R	GAGAGTAGTGGTGCTGGTGGTTGTG
85KO-F	TTCGCAACAGCGTGGTC
85KO-R	CCCGTCCGAGGAAAGAGT
Nsil-3-F	GGTCGGCATCTACTCTATTTCT
Nsil-3-R	GGTTCATTTAGGCAACTGGTC
85promoter-F	CCGGGTACCGAGCTCGAATTCTTGGGAGTGATAAAGGGCACC
85promoter-F	GCGCATGGTTAAGGCTTTGTTGTGTTGATTG
85hb-F	ACAAAGCCTTAACCATGCGCGCCTCCGTCCTC
85hb-R	ACTCATGAGAGTAGTGGTGCTGGTGGTTG
GFP-F	GCACCACTACTCTCATGAGTAAAGGAGAAGAACTTTTCACTG
GFP-R	CCCCTTTGTATAGTTCATCCATGCCATGT
85terminator-F	GGATGAACTATACAAAGGGGTTTGAAAACTCTTTCCTCG
85terminator-R	TATGGAGAAACTCGAGAATTCCTTCCAGGAGTATTCTTTGCAG
HBAtg8-F	TTTTACAAATACAAATACATACTAAGGGTTTCTTATATG
HBAtg8-R	TCGCTATTACGCCAGCTG
85-F	ATGCGCGCCTCCGTCCTCATC
85-R	GAGAGTAGTGGTGCTGGTGGTTGTG
G418-F	ATGAGTAAAGGAGAAGAACTTTTCACTG
G418-R	TTTGTATAGTTCATCCATGCCATGT

Tab.2 Disease grading criteria for Arabidopsis thaliana inoculated with Colletotrichum higginsianum

病情指数 Disease level	症状 Symptom
0	叶片无病斑
1	发病面积未超过叶片面积的1/3
2	发病面积超过叶片面积的1/3,但未及叶片面积的 2/3
3	发病面积超过叶片面积的 2/3

Fig.1 Signal peptide prediction of effector ChEP085 of Colletotrichum higginsianum in SingnalP 4.1

Fig.2 Expression patterns of effector gene ChEP085 of Colletotrichum higginsianum

Fig.3 The subcellular localization of effector ChEP085 of Colletotrichum higginsianum in Nicotiana benthamiana
A.The localization of empty vector pBinceGFP under fluorescence microscope;B.The localization of pBinceGFP-ChEP085;C.The localization of pBinceGFP-ChEP085-noSP.

Fig.4 Effector ChEP085 of Colletotrichum higginsianum induced cell death in Nicotiana benthamiana
A.Phenotype observation of tobacco leaves 6 d after Agrobactrium tumefacien infiltration;B.Phenotypic observation of tobacco leaves after decolorization treatment.1.PVX;2.PVX-INF1;3.PVX-ChEP085;4.PVX-ChEP085-noSP.

Fig.5 Knockout strategy of gene ChEP085

Fig.6 PCR detection of effector gene ΔChEP085 knockout mutants
A.PCR products were amplified using primers 85-F/R;B.PCR products were amplified using primers Hyg-F/R;C.PCR products were amplified using primers 85ko-F/R.M.DL5000 Marker;1—4.ΔChEP085-1/-2/-6/-8 knockout mutants;5.ddH₂O;6.Wild type.

Fig.7 Southern Blotting analysis of the wild type and ΔChEP085 knockout mutants
1.Wild type;2.ΔChEP085-2 knockout mutant; 3.ΔChEP085-8 knockout mutant.

Fig.8 PCR validation of ΔChEP085 complementary mutants
A.PCR products were amplified using primers 85-F/R;B.PCR products were amplified using primers G418-F/R;M.DS2000 Marker;1.ddH₂O;2.Wild type;3.ΔChEP085 knockout mutant;4.Complementary vector p822-85hb;.5—9(M12,2-13,8-5,8-7,8-9).Complementary mutants.

Fig.9 Growing states of the wild type,ΔChEP085 mutants and CΔChEP085 complementary strains
A.Colony morphology on PDA medium;B.Comparison of colony diameter;Data are mean±s.Different letters indicate significant difference of P<0.05.The same as Fig.10—11.

Fig.10 Analysis of conidia production of wild type, ΔChEP085 mutants and CΔChEP085 conplementary strains

Fig.11 Pathogenicity assays of the wild type,ΔChEP085 mutants and CΔChEP085 complementary strains
A.Virulence of the wild type,ΔChEP085 knockout and CΔChEP085 complementary mutants was determined by spraying conidial suspensions onto Arabidopsis plants,inoculated plants were photographed at 5 days post-inoculation;B.Barchart represents the calculated disease index of the seedlings infected by the indicated strains.

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