Metacaspase-like FpMca3 Involved in Pathogenicity of Fusarium pseudograminearum

doi:10.13560/j.cnki.biotech.bull.1985.2025-0423

Abstract

Abstract:

Objective To investigate the biological function of the cysteine protease-like FpMca3 in the pathogenicity process of Fusarium pseudograminearum may provide insights into the molecular mechanisms of pathogen virulence and potential drug targets for disease control. Method PCR and RT-PCR were adapted to amplify the FpMcas genes in F. pseudograminearum, and RT-qPCR was used to analyze the expressions of FpMca3 during pathogen infection stages. Bioinformatics tools were employed to analyze the domain structure, phylogenetic relationships, and sequence similarity of FpMca3. A FpMca3-knockout cassette was constructed using the split-PCR method and transformed into the wild-type strain of F. pseudograminearum via PEG-mediated protoplast transformation. The FpMca3-deleted mutants (Δfpmca3) were selected by hygromycin resistance screening and PCR verification, and the recombinant vector pKNTG-FpMca3 was constructed and introduced into the Δfpmca3 mutant to generate the complemented strain (Δfpmca3-C). Different strains of F. pseudograminearum were analyzed for growth on PDA medium, conidial production in CMC liquid culture, and germination rates in sterile water. Pathogenicity was tested by inoculating wheat coleoptiles and barley leaves with fungal mycelial plugs, and wheat crown rot was evaluated in pot experiments. Prokaryotic expression and protein purification were performed to examine the integrity of the FpMca3 protein. Result Genome analysis of F. pseudograminearum initially identified four potential metacaspase proteins containing Peptidase_C14 domains, but only three genes (FpMca1, FpMca2, and FpMca3) were successfully amplified with their complete gene and open reading frame sequences. Among them, FpMca1 and FpMca2 were type I metacaspases conserved in fungi, while FpMca3 resembled the metacaspase domain found in bacteria, representing a novel cysteine protease in fungi. FpMca3 was induced during the infection stages of F. pseudograminearum. Phenotypic characterization revealed no discernible differences in mycelial growth, conidial production and germination among the wild-type (WT), Δfpmca3 mutant, and Δfpmca3-C strains. However, the Δfpmca3 mutant demonstrated significantly reduced virulence compared to both the wild-type (WT) and complemented strains when inoculated on barley leaves and wheat coleoptiles and caused a marked decrease in wheat crown rot symptoms. The prokaryotically expressed FpMca3 presented two protein bands, suggesting that it possessed autocatalytic protease activity. Conclusion The metacaspase-like cysteine protease FpMca3 contributes to the virulence of F. pseudograminearum and has autocatalytic activity.

Key words: Fusarium pseudograminearum, metacaspase, FpMca3, virulence

CHEN Lin-lin, SU Zeng-qing, JI Xiao-ya, LIU Xin-yue, LIU Jia-yao, ZHANG Shi-yu, XING Xiao-ping, LI Hong-lian. Metacaspase-like FpMca3 Involved in Pathogenicity of Fusarium pseudograminearum[J]. Biotechnology Bulletin, 2025, 41(11): 272-281.

Figures/Tables 8

Table 1 Sequences of primers used in this study

Primer	Primer sequence （5′-3′）
FpMca1-F	ATGTCGCAATACCCAGGC
FpMca1-R	CTAATACTTGGTCAATGGC
FpMca2-F	ATGTCCTACTTTCCAGGTC
FpMca2-R	CATAACAAACAAGAGGTCGG
FpMca3-F	ATGTCCAACAAGGCCGTC
FpMca3-R	TCACACATTCGTAGGTACC
FpMca4-F	ATGACAAACCAACCGACTCATC
FpMca4-R	CTAAAGTACCGGTATCGAGGT
F1	ACAAGCGAATGCGAGTCGGTCAG
R1	TTGACCTCCACTAGCTCCAGCCAAGCCGATTGGTCTGACCATGTAAT
F2	ATAGAGTAGATGCCGACCGCGGGTTCAATTAATTGCAGAATTCTC
R2	TACTTCCAAAGTATATCACTGC
UP-F	GAGTGTGAGAGGATGTTGTC
H855R	GCTGATCTGACCAGTTGC
H856F	GTCGATGCGACGCAATCGT
Down-R	GCTGAATATCCTTTGCTGTTG
H850F	TTCCTCCCTTTATTTCAGATTCAA
H852R	ATGTTGGCGACCTCGTATTGG
G-F	CTAGCAATATCACCCTCATC
G-R	GGAGACTTTGGCAGTGTTC
Com-F	CTATAGGGCGAATTGGGTACCGAATTGATCTGTATACTGTAG
Com-R	GCAGGCATGCAAGCTTATCGATCACATTCGTAGGTACCATTTC
RT-F	GAGCTGGTCAACAATGGA
RT-R	CTTGTAGCCCCTTGAGTTC
FpTEF1-RTF	TCACCACTGAAGTCAAGTCC
FpTEF1-RTR	ACCAGCGACGTTACCACGTC

Fig. 1 Cloning and analysis of FpMcasA: Cloning of FpMca1-4 in F. pseudograminearum. B: Protein domains of FpMcas. C: Phylogenetic tree of FpMcas. D: Sequence analysis of the P20 subunit

Fig. 2 Expression analysis of FpMca3 during the infection stages of F. pseudograminearumThe expression of FpMca3 in mycelia was arbitrarily set as 1. Bars denote standard errors from four repeated experiments. IF18h, IF36h, IF2d, IF3d, IF5d, and IF7d represent 18 h, 36 h, 2 d, 3 d, 5 d, and 7 d after inoculation of wheat (Aikang 58) coleoptiles with the wild-type mycelium of F. pseudograminearum, respectively

Fig. 3 Generation and growth phenotype determination of FpMca3-knockout mutantA: PCR verification of the FpMca3-knockout mutants and FpMca3-complemented strains. B: Growth assay of the FpMca3-knockout mutant. The upper row shows the reverse side of the colonies, while the lower row displays the front side. C: Measurement of colony diameter. Lowercase letters indicate data difference at the P<0.05 level

Fig. 4 Conidial production and germination assaysA: Morphology of conidia and germination of the wild-type, Δfpmca3 and Δfpmca3-C F. pseudograminearum. B: The conidia quantity of the wild-type, Δfpmca3 and Δfpmca3-C strains. C: Germination rates of the wild-type, Δfpmca3 and Δfpmca3-C strains. Lowercase letters indicate data difference at the P<0.05 level

Fig. 5 Pathogenicity assay of F. pseudograminearum on barley leaves and wheat coleoptilesA: Phenotypes of lesions on barley leaves inoculated with the wild-type, Δfpmca3 and Δfpmca3-C F. pseudograminearum. B: Diameters of lesions on barley leaves measured at 4 d after inoculation. C: Phenotypes of lesions on wheat coleoptiles inoculated with the wild-type, Δfpmca3 and Δfpmca3-C strains. D: Diameters of lesions on wheat coleoptiles measured at 4 d after inoculation. ** P<0.01

Fig. 6 Pathogenicity assay of F. pseudograminearum in pot trialsA: Wheat growth induced by F. pseudograminearum infection. B: Crown rot disease index (DSI) in wheat seedlings infected with different F. pseudograminearum strains. ** P<0.01

Fig. 7 Detection of prokaryotically expressed FpMca3 proteinA: IPTG-induced expression of FpMca3 in prokaryotic cells. The black arrow marks the position of MBP protein expressed by the pMalc2x empty vector control. The red arrows mark the FpMca3-MBP fusion protein expressed in the recombinant strain. B: Purification of FpMca3-MBP. 1: Unpurified protein. 2: Supernatant (post-binding, CK1). 3: Supernatant after washing with MBP buffer (CK2). 4: Protein after eluted with PBS

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