Analysis of the Function of Pg&#x003B2;Glu4 Gene to Regulate Pathogenicity in Pyrenophora graminea

doi:10.7668/hbnxb.20195746

Abstract

Abstract:

To investigate the function of the β-glucosidase(βGlu)gene PgβGlu4 from Pyrenophora graminea,which previous studies found to be highly expressed during the infection stage,we constructed a subcellular localization vector of PCE2-EGFP-PgβGlu4 and transformed rice protoplasts,observed the fluorescence distribution and analyzed its location of existence.Simultaneously,the PgβGlu4 gene RNAi vector was constructed,and QWC protoplasts were prepared by CaCl₂-PEG4000 mediated method for genetic transformation.The function of PgβGlu4 gene was studied by detecting the vegetative growth and pathogenicity of the RNAi mutants.Phylogenetic analysis of PgβGlu4 and other homologous proteins from different pathogens showed that PgβGlu4 had a closer evolutionary relationship with that from Pyrenophora tritici-repentis.The subcellular localization results showed that PgβGlu4 was mainly localized in the nucleus and cell membrane.Four PgβGlu4 gene RNAi mutants were verified by hygromycin.qRT-PCR analysis showed that the expression of PgβGlu4 gene in four RNAi mutants decreased by 66.31%,68.60%,54.37% and 69.89%,respectively,compared with the wild isolate.The colony diameter was smaller than that of the wild isolate,and their incidence rate was reduced by 56.69,52.76,47.43,and 53.30 percentage points.After infection with the mutant strain of RNAi-PgβGlu4,the relative chlorophyll content in barley leaves ranged from 30.3 to 35.0,which was significantly higher than that of the wild-type group.The effect of PgβGlu4 gene silencing on the height of barley plants before and after infection was significant compared with that of the wild-type.The results indicated that the PgβGlu4 gene was involved in the regulation of the growth,development,and pathogenicity of Pyronophora graminea.

Key words: Pyrenophora graminea, β-glucosidase, RNA interference(RNAi), Pathogenicity

CLC Number:

S435.123

YANG Wenjuan, QI Tiantao, WANG Yanting, MENG Yaxiong, WANG Juncheng, WANG Huajun, SI Erjing. Analysis of the Function of PgβGlu4 Gene to Regulate Pathogenicity in Pyrenophora graminea[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 188-197. doi: 10.7668/hbnxb.20195746.

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http://www.hbnxb.net/EN/Y2025/V40/I5/188

Figures/Tables 11

Tab.1 Primers sequences used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
PgβGlu4-qRT-F	GAGCACCCCACACTTTACGA	实时荧光定量PCR
PgβGlu4-qRT-R	AAAGTACGGGCGGTTGTTGA
EGFP-PgβGlu4-F	TCATTTGGAGAGAACACGGGGGACTCTAGAATGCCACAAA	靶位点测序
	CACCACCAGCCACCTATCCTCGCCCCGACTTCCTTCG
EGFP-PgβGlu4-R	TTGCTCACCATAAGCTTGTCGACGGAGCTCTTATGCATCTG
	CGCATACGTTTCCATGGCATTTTCAACAACCTTCTTCACCTC
PgβGlu4-KpnⅠ-F	GG GGTACCCCACAAGGCCTGTAAACGAT	干扰片段克隆
PgβGlu4-BglⅡ-R	GA AGATCTTTTCCATGGCATTTTCAACA
PgβGlu4-XhoⅠ-F	CCG CTCGAGCCACAAGGCCTGTAAACGAT
PgβGlu-Hind Ⅲ-R	CCC AAGCTTTTTCCATGGCATTTTCAACA
HygB-F	ATGAAAAAGCCTGAACTCAC	干扰菌株筛选
HygB-R	CTATTCCTTTGCCCTCGGA
PgActin-F	TGTTGACATGGCTGGTCGTGATC	内参基因引物
PgActin-R	TCGGCGGTGGTGGAGAAGG

Fig.1 Schematic diagram of PgβGlu4 gene interference vector of Pyrenophora graminea

Fig.2 Phylogenetic tree of PgβGlu4 protein from Pyrenophora graminea and homologous proteins in other phytopathogenic

Fig.3 Subcellular localization plasmid and double enzyme digestion electrophoresis map of PgβGlu4 protein of Pyrenophora graminea 1.Plasmid of subcellular localization vector of PgβGlu4; 2.Double enzyme digestion electrophoresis;M.DL12000 DNA Marker.

Fig.4 The subcellular localization of PgβGlu4 protein of Pyrenophora graminea in rice cells From left to right are the superimposed photos of green fluorescent protein(GFP),bright field(DIC)and three channels(Merged)of empty vector EGFP and target gene PgβGlu4,respectively.

Fig.5 Gel electrophoresis image of double-digestion validation of interference vector pSilent-1∶PgβGlu4 A.Verification of interference fragment of PgβGlu4 gene of Pyrenophora graminea:1,2.Negative control ddH₂O,3.Interference fragment PgβGlu4-1,4.Interference fragment PgβGlu4-2,M.DL2000 DNA Marker;B.Double-digestion validation of interference vector pSilent-1∶PgβGlu4:1.Interference carrier pSilent-1∶PgβGlu4-1,2.Interference carrier pSilent-1∶PgβGlu4-2,M.DL2000 DNA Marker.

Fig.6 PCR verification of hygromycin resistance of PgβGlu4 gene RNAi mutants of Pyrenophora graminea M.DL2000 DNA Marker;1.PgβGlu4-RNAi-1;2.PgβGlu4-RNAi-2;3.PgβGlu4-RNAi-3;4.PgβGlu4-RNAi-4;5.ddH₂O;6.Wild strain;7.Empty carrier(pSilent-1).

Fig.7 Relative expression of PgβGlu4 RNAi mutants of Pyrenophora graminea Analysis of variance(ANOVA)was used for the differences test between different groups,and different lower case letters represented significant differences(P<0.05).The same as Fig.8—10.

Fig.8 Growth morphology and rate of wild strain and PgβGlu4 RNAi mutants of Pyrenophora graminea in PDA medium A.The apparent morphological diagram of Pyrenophora graminea;B.The microscopic structure diagram of mycelium of Pyrenophora graminea;C.The growth rates of wild-type QWC,interfering mutant strains were tested in PDA medium for 7 days;D.The colony diameter of each strain on the seventh day of inoculation in PDA medium.

Fig.9 Analysis pathogenicity of Pyrenophora graminea PgβGlu4 RNAi mutant strains on barley A.The potted symptoms of diseased barley plants infected with wild-type strain,PgβGlu4-RNAi interfering mutant strains at 20 days post-inoculation;B.Incidence statistics of barley infected by Pyrenophora graminea wild-type strain and PgβGlu4-RNAi mutant strains;C.Disease index of barley infected by Pyrenophora graminea wild-type strain and PgβGlu4-RNAi mutant strains;D.Height of barley leaves infected with wild-type strain, PgβGlu4-RNAi mutant strains after 20 days of infection;E.Relative chlorophyll content of barley leaves infected with wild-type strain,PgβGlu4-RNAi mutant strains after 20 days of infection.

Fig.10 The trypan blue staining condition of symptoms of barley leaves infected by wild type strain and PgβGlu4 gene RNAi mutants of Pyrenophora graminea A.Trypan blue staining of diseased barley leaves after 20 days of infection with wild-type strain,PgβGlu4-RNAi mutant strains;B.Lesion length of barley leaves infected with wild-type strain,PgβGlu4-RNAi mutant strains after 20 days of infection.

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