Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (16): 3440-3450.doi: 10.3864/j.issn.0578-1752.2021.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Virulence Analysis of CAP Superfamily Genes in Valsa mali

WANG ChengLi(),YIN ZhiYuan,NIE JiaJun,LIN YongHui,HUANG LiLi()   

  1. College of Plant Protection, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
  • Received:2020-11-08 Accepted:2020-12-17 Online:2021-08-16 Published:2021-08-24
  • Contact: LiLi HUANG E-mail:wein599@163.com;huanglili@nwsuaf.edu.cn

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Abstract:

【Objective】CAP (Cysteine-rich secretory protein, Antigen 5 and Pathogenesis related protein 1) superfamily proteins widely exist in fungi, bacteria, animal and plant. This kind of proteins participates in the pathogenic process of the pathogen. The purpose of this study was to identify the CAP superfamily genes in Valsa mali and clarify their virulence roles.【Method】BLASTP was used to retrieve genes with the conserved CAP domains in the whole genome of V. mali. PCR amplification and gel electrophoresis detection were carried out with specific primers. Bioinformatics software and online databases were used for protein sequence characterization and phylogenetic analysis. RT-qPCR was used to analyze the gene expression profiles. Double-joint PCR was used to construct gene knocking-out cassettes, PEG-mediated protoplast transformation was used to obtain knocking-out mutants and complementation strains. To obtain gene knockout mutants, geneticin (G418) was used as a selection marker, and transformants were validated by four pairs of primers. To obtain gene complementation transformants, hygromycin (HPH) was used as a selection marker. The vegetative growth of these strains was determined by cultivation on PDA medium, and the virulence of these strains was verified by inoculation on apple twigs.【Result】Three CAP superfamily genes were identified in V. mali, named VmPR1a, VmPR1b and VmPR1c, respectively. The three CAP proteins all contain four conserved regions, including a N-terminal signal peptide, a N-terminal extension region (NTE), a CAP domain and a C-terminal extension region (CTE). Phylogenetic analysis showed that the three proteins belonged to different clades. VmPR1a was clustered in clade2 and closely related to Neurospora crassa CAP protein. VmPR1b was clustered in clade3 and closely related to CAP proteins of Fusarium spp.. VmPR1c was clustered in clade1 and also closely related to N. crassa CAP protein. RT-qPCR analysis showed that VmPR1a, VmPR1b and VmPR1c were significantly up-regulated during early stages of infection (6 h and 12 h). VmPR1a, VmPR1b and VmPR1c knockout mutants (ΔVmPR1a-7/23, ΔVmPR1b-20/31 and ΔVmPR1c-26/40, respectively) were obtained, and all mutants showed no apparent alteration in filamentous growth compared with that of the wild-type strain. The virulence of ΔVmPR1b-20/31 was not obviously influenced. However, the virulence of ΔVmPR1a-7/23 and ΔVmPR1c-26/40 was significantly reduced compared with that of the wild-type strain. Moreover, the virulence of gene complementation strains (VmPR1a/C and VmPR1c/C) was restored to comparable level as that of the wild-type. 【Conclusion】Three CAP super-family genes were identified in V. mali (VmPR1a, VmPR1b and VmPR1c), and VmPR1a and VmPR1c are virulence factors of V. mali.

Key words: Valsa mali, CAP protein, gene identification, gene knockout, virulence

Table 1

Primers information used in this study"

引物 Primer 序列 Sequence (5′→3′)
VmPR1a-F ggggactcttgaccatggtaATGAAGCCCGCTC
VmPR1a-R ctcaccatcctaggactagtTAACGCCAGAAACAGTACCA
VmPR1b-F ggggactcttgaccatggta ATGCACTTCTCAACTCGTCC
VmPR1b-R ctcaccatcctaggactagtAGCAGAGGTTGGGGTCTC
VmPR1c-F ggggactcttgaccatggta ATGAAGTCATCATCGCTCTT
VmPR1c-R ctcaccatcctaggactagtTACGCTGACGTTCACGGA
pCA1302-F/R TAAGGGATGACGCACAAT/CGGACACGCTGAACTTGT
qRT-VmPR1a-F/R AGTCGCCAGACTCCGGATCCG/GACCAACTGCGTGAAGTGGC
qRT-VmPR1b-F/R ACATCCTCGTCTGCTGCTGCTG/GGTCTCGCTAACTCCAGAACGA
qRT-VmPR1c-F/R CATGACCTACGGTGACACGTA/GAGTCAGCGTCAGTAGAGGC
G6PDH-F/R TCAGAACAAGTTCGAGGGCGACAA/TGAGGGCAATAGAGGGCTTGTTCA
VmPR1a-5F/6R ACGCTTTCATCCAGACTTTT/CCTTCTCCTTGTGGCTTTGT
VmPR1b-5F/6R CACTTCTCAACTCGTCCCG/ACCGTCCGAAGCAATACC
VmPR1c-5F/6R GAGGTTACGACCGTTGAGAC/CAGTGAAAAGGGGATGGC
Neo-F/R CACAGCACCGACCACAAA/CCAGCAGTAGACACTTGGAATC
PDL2-VmPR1a-F cgactcactatagggcgaattgggtactcaaattggttataaACGCTTTCATCCAGACTTTT
PDL2-VmPR1a-R ccaccccggtgaacagctcctcgcccttgctcacctcgagCCTTCTCCTTGTGGCTTTGT
PDL2-VmPR1c-F cgactcactatagggcgaattgggtactcaaattggttataaCGATTTGGGCAACTTGAG
PDL2-VmPR1c-R ccaccccggtgaacagctcctcgcccttgctcacctcgagGCGATAGCCAGAAAACATAG

Fig. 1

Construction of gene knocking-out cassettes"

Fig. 2

Gel electrophoresis detection of VmPR1a, VmPR1b and VmPR1c"

Fig. 3

Phylogenetic analysis of VmPR1a, VmPR1b and VmPR1c The maximum-likelihood method was used for the construction of phylogenetic-tree. Bootstrap value supporting for each branch is indicated"

Fig. 4

Prediction of the signal peptide, conserved domain and active amino acid site in V. mali CAP superfamily proteins"

Fig. 5

Relative expression level of VmPR1a, VmPR1b and VmPR1c at different infection stages of V. mali"

Fig. 6

PCR detection of mutants and complementation strains"

Fig. 7

Colony morphology and colony diameter of the wild-type, mutants and complementation strains inoculated on PDA medium"

Fig. 8

Pathogenicity detection of the VmPR1a, VmPR1b and VmPR1c mutants"

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