Functional Analysis of the Type III Secreted Effector Gene aop2 in Acidovorax citrulli

doi:10.13560/j.cnki.biotech.bull.1985.2022-1303

Abstract

Abstract:

Type III secreted effectors are the key pathogenic factors secreted by Acidovorax citrulli, the pathogen of bacterial fruit blotch（BFB）. To identify the T3SE gene aop2 with GNAT（Gcn5-related N-acetyltransferase）superfamily domain, which is specific for Acidovorax citrulli, and to analyze the way that its encoded protein affects plant immunity, will lay a foundation for further understanding the role of this gene in pathogen pathogenesis. Bioinformatics was applied to analyze the sequence characteristics. Fluorescence quantitative PCR was used to analyze the expression regulation of aop2 and its relationship with the expression of disease-resistant genes. Gene mutation and gene function complementation was to analyze the gene function, including pathogenicity and host reactive oxygen species accumulation. Transient expression technique was used to investigate the ability of Aop2 inhibitory elicitor to induce necrosis and its subcellular localization. There was a core gene binding site of Type III secretion system（T3SS）in the promoter region of aop2 gene, and there was no signal peptide or transmembrane helical region in the encoded protein, and there was a GNAT family acetyltransferase domain but no homologous protein. The expression of aop2 gene in hrpG/hrpX mutant of T3SS significantly decreased. The mutant with aop2 gene deletion decreased the pathogenicity of host cucumber, but increased the accumulation of reactive oxygen species in cucumber cotyledon. Aop2 can be localized to whole tobacco cells and inhibited programmed cell death（PCD）induced by NIP. The expression of Aop2 enhanced the expression of genes involved in SA and JA signaling pathways triggered by pathogen-related molecular patterns in tobacco leaves. The results showed that Aop2 was a specific T3SE containing GNAT domain in A. citrulli. Aop2 played the function of virulence factor in the interaction with cucumber and was involved in the regulation of plant cell death and PTI and phytohormone-related disease resistance in the interaction with tobacco.

Key words: type III secreted effectors, aop2, virulence factor, cell death, acetyltransferase, PTI

CHEN Bao-qiang, LI Ying-ying, MA Bo-ya, ROUZHAGULI Malike, YOULITUZI Naibi, SONG Jin-di, LIU Jun, WANG Xi-dong. Functional Analysis of the Type III Secreted Effector Gene aop2 in Acidovorax citrulli[J]. Biotechnology Bulletin, 2023, 39(6): 286-297.

Figures/Tables 10

References 40

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菌株和质粒 Strain and plasmid	名称 Name	特性 Characteristic	来源Source
菌株 Strain	FC440（WT）	Amp^R; wild type	Our laboratory
	FC440（∆aop2）	Amp^R; Kan^R; FC440 mutant defective in aop2	This study
	FC440（∆aop2-aop2）	Amp^R; Kan^R; Gm^R; FC440（∆aop2）complemented with the fragment of aop2 gene expressed by vector pBBR1MCS-5	This study
	FC440（∆hrpG）	AmpR; Kan^R; FC440 mutant defective in hrpG	[14]
	FC440（∆hrpX）	Amp^R; Kan^R; FC440 mutant defective in hrpX	[14]
	Trans T1（pBBR1MCS-5- aop2）	Gm^R; E. coli TransT1 strain containing vector pBBR1MCS-5-aop2	This study
	S17-1（pBBR1MCS-5- aop2）	Gm^R; E. coli S17-1 strain containing vector pBBR1MCS-5aop2	This study
	GV3101	Rif^R; wild type	Our laboratory
	GV3101（pAPK-aop2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-aop2	This study
	GV3101（pAPK-GFP）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-GFP	[15]
	GV3101（pBINGFP2-aop2）	Kan^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2-aop2	This study
	GV3101（pBINGFP2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2	[15]
	GV3101（BAX）	Kan^R; Rif^R; Gm^R; GV3101 strain containing BAX	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（Avh241）	Kan^R; Rif^R; Gm^R; GV3101 strain containing Avh241	[15]
	GV3101（INF1）	Kan^R; Rif^R; Gm^R; GV3101 strain containing INF1	[15]
质粒 Plasmid	pMD19-T	Amp^R; Cloning vector	TaKaRa
	pMD19-T- aop2	Amp^R; pMD19-T vector containing a 438-bp fragment with the aop2 gene	This study
	pK19mob2ΩHMB-aop2	Kan^R; pK19mob2ΩHMB vector containing a 301-bp fragment with the aop2 gene	This study
	pBBR1MCS-5-aop2	Gm^R; pBBR1MCS-5 vector containing aop2 gene; used to complement FC440（∆aop2）	This study
	pKannibal-aop2	Kan^R; pKannibal vector containing a 438-bp fragment with the aop2 gene	This study
	pAPK-aop2	Spec^R; pART27 vevtor containing pKannibal-aop2	This study
	pBINGFP2-aop2	Kan^R; pBINGFP2 vector containing a 438-bp fragment with the aop2 gene	This study

菌株和质粒 Strain and plasmid	名称 Name	特性 Characteristic	来源Source
菌株 Strain	FC440（WT）	Amp^R; wild type	Our laboratory
	FC440（∆aop2）	Amp^R; Kan^R; FC440 mutant defective in aop2	This study
	FC440（∆aop2-aop2）	Amp^R; Kan^R; Gm^R; FC440（∆aop2）complemented with the fragment of aop2 gene expressed by vector pBBR1MCS-5	This study
	FC440（∆hrpG）	AmpR; Kan^R; FC440 mutant defective in hrpG	[14]
	FC440（∆hrpX）	Amp^R; Kan^R; FC440 mutant defective in hrpX	[14]
	Trans T1（pBBR1MCS-5- aop2）	Gm^R; E. coli TransT1 strain containing vector pBBR1MCS-5-aop2	This study
	S17-1（pBBR1MCS-5- aop2）	Gm^R; E. coli S17-1 strain containing vector pBBR1MCS-5aop2	This study
	GV3101	Rif^R; wild type	Our laboratory
	GV3101（pAPK-aop2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-aop2	This study
	GV3101（pAPK-GFP）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-GFP	[15]
	GV3101（pBINGFP2-aop2）	Kan^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2-aop2	This study
	GV3101（pBINGFP2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2	[15]
	GV3101（BAX）	Kan^R; Rif^R; Gm^R; GV3101 strain containing BAX	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（Avh241）	Kan^R; Rif^R; Gm^R; GV3101 strain containing Avh241	[15]
	GV3101（INF1）	Kan^R; Rif^R; Gm^R; GV3101 strain containing INF1	[15]
质粒 Plasmid	pMD19-T	Amp^R; Cloning vector	TaKaRa
	pMD19-T- aop2	Amp^R; pMD19-T vector containing a 438-bp fragment with the aop2 gene	This study
	pK19mob2ΩHMB-aop2	Kan^R; pK19mob2ΩHMB vector containing a 301-bp fragment with the aop2 gene	This study
	pBBR1MCS-5-aop2	Gm^R; pBBR1MCS-5 vector containing aop2 gene; used to complement FC440（∆aop2）	This study
	pKannibal-aop2	Kan^R; pKannibal vector containing a 438-bp fragment with the aop2 gene	This study
	pAPK-aop2	Spec^R; pART27 vevtor containing pKannibal-aop2	This study
	pBINGFP2-aop2	Kan^R; pBINGFP2 vector containing a 438-bp fragment with the aop2 gene	This study

Primer name	Sequnece（5'-3'）	Source
aop2-F	CGGAATTCTGCTGGAGGTCCTGCTGT（EcoR I）	This study
aop2-R	CCAAGCTTACTTCCATCGCATCGTCC（Hind III）	This study
aop2-QF	CCCTCGAGTGGCAACGATGTTTCCCC（Xho I）	This study
aop2-QR	GCTCTAGAGCGACAGCGACGGCTGAG（Xba I）	This study
AAC-1	GACCAGCCCACAACTGGGAC	[16]
AAC-2	CTGCCGCACTCCAGCGA	[16]
aop2-CF	TTTGGAGAGGACACGCTCGAGATGACAGACAGACTCAGCCGGC（Xho I）	This study
aop2-CR	TCATTAAAGCAGGACTCTAGATCAGTTCACCGTTGACGACGC（Xba I）	This study
aop2-DF	AGAGGATCCGTCGACCCCGGGATGAGCCTGCAGAACGCCA（Sma I）	This study
aop2-DR	CTGTACAAGGGTACCCCCGGGTCAGGCGTCCAGGGGCAG（Sma I）	This study
RT-aop2-F	CCTGGCTGGCGGACAAGT	This study
RT-aop2-R	CTGCCCGAAGAACTGCGA	This study
rpoB-F	GCGACAGCGTGCTCAAAGTG	[17]
rpoB-R	GGCCTTCGTTGGTGCGTTTCT	[17]
NbPti5-F	CCTCCAAGTTTGAGCTCGGATAGT	[18]
NbPti5-R	CCAAGAAATTCTCCATGCACTCTGTC	[18]
NbAcre31-F	AATTCGGCCATCGTGATCTTGGTC	[18]
NbAcre31-R	GAGAAACTGGGATTGCCTGAAGGA	[18]
NbGras2-F	TACCTAGCACCAAGCAGATGCAGA	[18]
NbGras2-R	TCATGAGGCGTTACTCGGAGCATT	[18]
NbEF1α-F	AAGGTCCAGTATGCCTGGGTGCTTGAC	[18]
NbEF1α-R	AAGAATTCACAGGGAC AGTTCCAATACCA	[18]
NbWRKY7-F	CACAAGGGTACAAACAACACAG	[19]
NbWRKY7-R	GGTTGCATTTGGTTCATGTAAG	[19]
NbWRKY8-F	AACAATGGTGCCAATAATGC	[19]
NbWRKY8-R	TGCATATCCTGAGAAACCATT	[19]
NbPR2b-F	TCCAACTTGGAATCAAAGGG	[20]
NbPR2b-R	GTGGACACTATACTCAGGTG	[20]
NbLOX-F	AAAACCTATGCCTCAAGAAC	[20]
NbLOX-R	ACTGCTGCATAGGCTTTGG	[20]
NbEFR1-F	GCTCTTAACGTCGGATGGTC	[20]
NbEFR1-R	AGCCAAACCCTAGCTCCATT	[20]
NbGAPDH-F	AGCTCAAGGGAATTCTCGATG	[21]
NbGAPDH-R	AACCTTAACCATGTCATCTCCC	[21]

Primer name	Sequnece（5'-3'）	Source
aop2-F	CGGAATTCTGCTGGAGGTCCTGCTGT（EcoR I）	This study
aop2-R	CCAAGCTTACTTCCATCGCATCGTCC（Hind III）	This study
aop2-QF	CCCTCGAGTGGCAACGATGTTTCCCC（Xho I）	This study
aop2-QR	GCTCTAGAGCGACAGCGACGGCTGAG（Xba I）	This study
AAC-1	GACCAGCCCACAACTGGGAC	[16]
AAC-2	CTGCCGCACTCCAGCGA	[16]
aop2-CF	TTTGGAGAGGACACGCTCGAGATGACAGACAGACTCAGCCGGC（Xho I）	This study
aop2-CR	TCATTAAAGCAGGACTCTAGATCAGTTCACCGTTGACGACGC（Xba I）	This study
aop2-DF	AGAGGATCCGTCGACCCCGGGATGAGCCTGCAGAACGCCA（Sma I）	This study
aop2-DR	CTGTACAAGGGTACCCCCGGGTCAGGCGTCCAGGGGCAG（Sma I）	This study
RT-aop2-F	CCTGGCTGGCGGACAAGT	This study
RT-aop2-R	CTGCCCGAAGAACTGCGA	This study
rpoB-F	GCGACAGCGTGCTCAAAGTG	[17]
rpoB-R	GGCCTTCGTTGGTGCGTTTCT	[17]
NbPti5-F	CCTCCAAGTTTGAGCTCGGATAGT	[18]
NbPti5-R	CCAAGAAATTCTCCATGCACTCTGTC	[18]
NbAcre31-F	AATTCGGCCATCGTGATCTTGGTC	[18]
NbAcre31-R	GAGAAACTGGGATTGCCTGAAGGA	[18]
NbGras2-F	TACCTAGCACCAAGCAGATGCAGA	[18]
NbGras2-R	TCATGAGGCGTTACTCGGAGCATT	[18]
NbEF1α-F	AAGGTCCAGTATGCCTGGGTGCTTGAC	[18]
NbEF1α-R	AAGAATTCACAGGGAC AGTTCCAATACCA	[18]
NbWRKY7-F	CACAAGGGTACAAACAACACAG	[19]
NbWRKY7-R	GGTTGCATTTGGTTCATGTAAG	[19]
NbWRKY8-F	AACAATGGTGCCAATAATGC	[19]
NbWRKY8-R	TGCATATCCTGAGAAACCATT	[19]
NbPR2b-F	TCCAACTTGGAATCAAAGGG	[20]
NbPR2b-R	GTGGACACTATACTCAGGTG	[20]
NbLOX-F	AAAACCTATGCCTCAAGAAC	[20]
NbLOX-R	ACTGCTGCATAGGCTTTGG	[20]
NbEFR1-F	GCTCTTAACGTCGGATGGTC	[20]
NbEFR1-R	AGCCAAACCCTAGCTCCATT	[20]
NbGAPDH-F	AGCTCAAGGGAATTCTCGATG	[21]
NbGAPDH-R	AACCTTAACCATGTCATCTCCC	[21]