Functional Analysis of Pg03852 Gene in Pyrenophora graminea

doi:10.13560/j.cnki.biotech.bull.1985.2025-0741

Abstract

Abstract:

Objective To explore the function of gene Pg03852 in Pyrenophora graminea, thereby establishing a crucial theoretical foundation for studying the molecular interactions between this pathogen and its host plant. Method Based on transcriptomic data, the highly expressed gene Pg03852 during the host infection process of P. graminea was identified. Preliminary functional characterization of this gene was conducted by utilizing a combination of bioinformatic analyses, signal peptide activity assays, induction/suppression of immune responses, and the generation of RNA interference (RNAi) mutants followed by pathogenicity assessment. Result Bioinformatics analysis indicated that the Pg03852 gene encoded 276 amino acids, with a secondary structure predominantly composed of disordered coils accounting for 73.19%.Pg03852 was an unstable hydrophilic protein lacking transmembrane regions and conserved domains, but possessing a signal peptide and localized to the extracellular space. Signal peptide activity assays demonstrated that the Pg03852 signal peptide transformant grew normally on CMD-W and YPRAA media. TTC staining further confirmed the secretory function of this protein’s signal peptide. Results from immune response induction and inhibition experiments showed that Pg03852 did not induce cell necrosis but suppressed BAX-induced necrosis in plant tissues. Two RNAi mutants, Pg03852-RNAi-1 and Pg03852-RNAi-2, were obtained via PEG-mediated protoplast transformation. RT-qPCR results showed that Pg03852-RNAi-1 and Pg03852-RNAi-2 had expression reductions of 58.76% and 48.24% compared to the wild-type strain QWC, respectively, with disease incidence rates of 34.67% and 38.00% lower than the wild-type strain QWC. Both mutants also demonstrated significantly slower colony growth rates than the wild-type strain QWC (P<0.05). Histological observations revealed that hyphae in the RNAi mutants were denser, more convoluted, and had a slender morphology. Trypan blue staining and DAB staining results indicated that both the extent of cell death induced by infection with the interference mutants and the levels of reactive oxygen species accumulation were weaker than those observed in wild-type strain QWC. Conclusion Pg03852 is involved in the growth and development of the P. graminea and plays a critical role in regulating its pathogenicity.

Key words: Pyrenophora graminea, signal peptide, immune response, RNA interference, pathogenicity

MA Ru-qing, FENG Xue-qi, YANG Qing-lan, WANG Jun-cheng, MENG Ya-xiong, MA Xiao-le, LI Bao-chun, YAO Li-rong, WANG Hua-jun, SI Er-jing. Functional Analysis of Pg03852 Gene in Pyrenophora graminea[J]. Biotechnology Bulletin, 2026, 42(1): 262-270.

Figures/Tables 6

Table 1 Primer list

引物 Primer	序列 Sequence（5'-3'）	用途 Purpose
Pg03852-pSuC2-F	AATTCATGAAGGTCTCACAGTTTCTCACCTGCCTCATTGCCGCGCAAGCAATCACCGCCC	构建酵母表达载体
Pg03852-pSuC2-R	TCGAGGGCGGTGATTGCTTGCGCGGCAATGAGGCAGGTGAGAAACTGTGAGACCTTCATG
Pg03852-Cal I-F	CCCATCGATATGAAGGTCTCACAGTTTCT	干扰片段克隆
Pg03852-Sma I-R	TCCCCCGGGCATGAACACACCAGCAAGAA
Pg03852-Kpn I-F	GGGGTACCTCCTCTCATCGGACAACACA
Pg03852-Bgl II-R	GAAGATCTGTTCTCGTCACAGCCACAGA
Pg03852-Xho I-F	CCGCTCGAGTCCTCTCATCGGACAACACA
Pg03852-Hind III-R	CCCAAGCTTGTTCTCGTCACAGCCACAGA
HygB-F	ATGAAAAAGCCTGAACTCAC	潮霉素鉴定
HygB-R	CTATTCCTTTGCCCTCGGA
Pg03852-qPCR-F	AGCAACACCATACAGAGCCG	实时荧光定量PCR
Pg03852-qPCR-R	GAAGCGGTAGGGGTTGTTGT	实时荧光定量PCR
PgActin-F	TGTTGACATGGCTGGTCGTGATC	内参基因
PgActin-R	TCGGCGGTGGTGGAGAAGG	内参基因

Fig. 1 Activity assay of Pg03852 signal peptide

Fig. 2 Pg03852 induces/inhibits the death phenotypes of necrotic cells in Baccata nicotiana leaves

Fig. 3 Identification of thaumatin resistance in interfering strains (A) and detection of relative expression of the Pg03852 gene (B)A: DL 15000+2 000 DNA marker; lane 1 is the empty vector (pSilent-1); lane 2 is the wild strain QWC; lane 3-4 are the mutants Pg03852-RNAi-1 and Pg03852-RNAi-2. B. Relative expression of Pg03852. A t-test was used, and * indicates significant differences at the 0.05 levelA: Pg03852 effector protein alone was identified in this tobacco, BAX and INF1 were used as positive control, and GFP was used as negative control; B: Pg03852 effector protein co-expressed with BAX was identified in this tobacco, PVX: BAX+PVX:Avh240 injected tobacco leaves were used as positive control, and PVX: BAX+GFP was used as negative control

Fig. 4 Growth morphology, growth rate, and hyphal morphology observed after 7 d of inoculation on PDA mediumA: Growth morphology of wild-type QWC, interference mutant strains Pg03852-RNAi-1, and Pg03852-RNAi-2 after 7 d of inoculation on PDA medium. B: Observation of mycelium morphology. C: Strain growth rate

Fig. 5 Pathogenicity analysis and stainingA: Statistics of QWC and interference mutant incidence (P<0.05). B: Relative chlorophyll content of QWC and interference mutants (P<0.05). C: Staining results of QWC and interference mutants

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