多主棒孢菌CcTLS1对黄瓜的致病机理分析

doi:10.16420/j.issn.0513-353x.2021-1056

摘要/Abstract

摘要：

为了解多主棒孢菌CcTLS1基因对黄瓜棒孢叶斑病的致病机理，以多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104为研究对象，通过对CcTLS1在强致病力菌株中敲除、回补以及在弱致病力菌株中异源表达等方法，研究CcTLS1在多主棒孢菌对黄瓜致病过程中的作用。CcTLS1全长为1 222 bp，与GenBank中已注释的基因无序列相似性。与野生型菌株相比，CcTLS1基因敲除菌株对黄瓜的致病力明显下降，产孢量和产孢梗数量显著减少，菌丝变细易卷曲，隔膜减少，纤维二糖水解酶分泌显著降低；而CcTLS1回补菌株致病力、产孢量以及纤维二糖水解酶分泌得到恢复；与野生型菌株相比，CcTLS1异源表达菌株对黄瓜的致病力提升，菌丝粗壮且隔膜增多，纤维素酶及纤维二糖分解酶分泌显著增加。影响多主棒孢菌致病的蛋白激酶基因CCK1、产孢相关基因（Ccflbc、CcstuA）以及黑色素合成相关基因CcSCD1，在CcTLS1基因敲除菌株中的相对表达量显著下调。结果表明，CcTLS1的表达影响菌株产孢机制、菌丝生长、蛋白激酶表达以及黑色素合成，在多主棒孢菌致病中起重要作用。

关键词: 黄瓜, 棒孢叶斑病, 多主棒孢菌, CcTLS1, 产孢, 致病力, 转录表达

Abstract:

In order to understand the pathogenic mechanism of the pathogenicity-related gene CcTLS1 from Corynespora cassiicola，virulent strain HG141025224 and the low virulent strain HG15052104 were used as the research object. Through genetic transformation of CcTLS1 gene deletion and complement of virulent strain and heterologous expression of low virulent strain，to study the role of CcTLS1 gene in the pathogenicity of C. cassiicola. The full length of CcTLS1 is determined to be 1 222 bp，which has no sequence similarity with the annotated genes in GenBank. Compared with wild strain HG14102524，the CcTLS1 knockout mutant ΔCcTLS1 showed decreased virulence on cucumber plant significantly，the conidiation and conidiophores decreased，the hyphae became thin and easily friable，the septum decreased，and the secretion of cellobiohydrolase lowered. The pathogenicity，conidiation and cellobiohydrolase secretion of complement mutant cΔCcTLS1 were recovered. Compared with wild strain HG15052104，the CcTLS1 heterologous expression mutant AH3-23 showed improved virulence on cucumber plant significantly，hyphae were thick and the septum increased，and the secretion of cellulase and cellobiohydrolase was significantly augmented. The relative expression levels of protein kinase genes CCK1，conidiation-related genes（Ccflbc，CcstuA）and melanocyte formation related genes CcSCD1 were significantly down-regulated in CcTLS1 gene knockout mutant. The results showed that CcTLS1 gene affected sporulation mechanism，mycelia growth，expression of protein kinase and melanin synthesis，and played an important role in pathogenicity of C. cassiicola.

Key words: cucumber, target leaf spot, Corynespora cassiicola, CcTLS1, conidiation, pathogenicity, transcriptional expression

中图分类号:

S642.2

王泉城, 武军, 李磊, 石延霞, 谢学文, 李宝聚, 柴阿丽. 多主棒孢菌CcTLS1对黄瓜的致病机理分析[J]. 园艺学报, 2023, 50(3): 569-582.

WANG Quancheng, WU Jun, LI Lei, SHI Yanxia, XIE Xuewen, LI Baoju, CHAI Ali. Exploration on the Function of Pathogenicity-related Gene CcTLS1 in Corynespora cassiicola From Cucumber[J]. Acta Horticulturae Sinica, 2023, 50(3): 569-582.

图/表 12

表1 载体构建及突变菌株验证所用引物序列

Table 1 Primer sequences for vector construction and mutant validation

基因、载体 Gene，vector	引物 Primer	序列（5′-3′） Sequence	参考文献 Reference
CcTLS1	CcTLS1	F：ATGCGTTCCTCAATCATCATCGC
CcTLS1		R：TTACAATGGCTGTCGTACTGGG
pCAMBIA1300-ΔCcTLS1	CcTLS1-51	F：CCATGATTACGAATTCATCCGGTCTGTTGGGTCG
		R：AACCGCCTCTCCCCGGTACCGTTGTCAGTTGTGAGGAGTATT
	CcTLS1-31	F：AACAGCTCCCTCTAGAAGACCAAAAGAGCATTAGTTCATC
		R：CCAAGCTTGCATGCCTGCAGAGATCTCATTGGCAGCGTAAAAAC
	CcTLS1-h	F：GGGGTACCGGGGAGAGGCGGTTTGC
		R：GCTCTAGAGGGAGCTGTTGGCTGGC
pCAMBIA1300-cΔCcTLS1	CcTLS1-52	F：CCATGATTACGAATTCGCTCGGGATTTGGCACACG
		R：CTAGAGGATCCCCGGGTACCTTACAATGGCTGTCGTACTGGG
	CcTLS1-32	F：GTTCTTCTGAAGACCAAAAGAGCATTAGTTCATCA
		R：CAGTGCCAAGCTTGCATGCCTGCAGGAGCTTACTGGGGTCCGGG
	CcTLS1-g	F：GGTACCCGGGGATCCTCTAGAGTCGACAGAAGATGATATTGAAGG
		R：CTTTTGGTCTTCAGAAGAACTCGTCAAGAAGGC
pCAMBIA1300-h， pCAMBIA1300-heΔCcTLS1	CcITS-5	F：CCATGATTACGAATTCCTTAGATGTTCTGGGCCGCAC
		R：CTAGAGGATCCCCGGGTACCTACTTCCTCTAAATGACCGAGTTTG
	CcITS-3	F：AACAGCTCCCTCTAGAAAAGAAACCAACAGGGATTGCC
		R：GCAGGTCGACTCTAGAGGGAGCTGTTGGCTGGCTGG
	CcITS-h	F：ATTTAGAGGAAGTAGGTACCGGGGAGAGGCGGTTTGCG
		R：GCAGGTCGACTCTAGAGGGAGCTGTTGGCTGGCTGG
	CcTLS1-I	F：ATTTAGAGGAAGTAGGGCAGGTACTGGTTCGGATGT
		R：AACCGCCTCTCCCCGACAGCCTTTCGGCGGTTAGA
突变株验证 Mutant validation	FhphU	TGTCCTGCGGGTAAATAGC	Fu et al.，2012
	FhphL	TTGTTGGAGCCGAAATCC
	FrbU	CCTCTTCGCTATTACGCC
	FrbUC	GGCGTAATAGCGAAGAGG
	M13R	CAGGAAACAGCTATGACC	通用引物 Universal primer
	FhphLL	GGATTTCGGCTCCAACAA
	F5F	TTGTTGGAGCCGAAATCC
	F3F	AATTGCGATAACGAACGAGACC
	F4F2	GAATTAGCTGCGCTCATGCAC
	Neo-G418	F：GTCGACAGAAGATGATATTG	张晓珂，2015
		R：TCAGAAGAACTCGTCAAGAAGGCG	张晓珂，2015
	qSdhB	F：GGCGAAGTTGCCGTGAG
		R：CTTGGTGGTATCGGTGGG
	qCcTLS1	F：GACGAGGATGCTGATGAGGA
		R：TGTCGTACTGGGATGCTGTT

表1 载体构建及突变菌株验证所用引物序列

Table 1 Primer sequences for vector construction and mutant validation

基因、载体 Gene，vector	引物 Primer	序列（5′-3′） Sequence	参考文献 Reference
CcTLS1	CcTLS1	F：ATGCGTTCCTCAATCATCATCGC
CcTLS1		R：TTACAATGGCTGTCGTACTGGG
pCAMBIA1300-ΔCcTLS1	CcTLS1-51	F：CCATGATTACGAATTCATCCGGTCTGTTGGGTCG
		R：AACCGCCTCTCCCCGGTACCGTTGTCAGTTGTGAGGAGTATT
	CcTLS1-31	F：AACAGCTCCCTCTAGAAGACCAAAAGAGCATTAGTTCATC
		R：CCAAGCTTGCATGCCTGCAGAGATCTCATTGGCAGCGTAAAAAC
	CcTLS1-h	F：GGGGTACCGGGGAGAGGCGGTTTGC
		R：GCTCTAGAGGGAGCTGTTGGCTGGC
pCAMBIA1300-cΔCcTLS1	CcTLS1-52	F：CCATGATTACGAATTCGCTCGGGATTTGGCACACG
		R：CTAGAGGATCCCCGGGTACCTTACAATGGCTGTCGTACTGGG
	CcTLS1-32	F：GTTCTTCTGAAGACCAAAAGAGCATTAGTTCATCA
		R：CAGTGCCAAGCTTGCATGCCTGCAGGAGCTTACTGGGGTCCGGG
	CcTLS1-g	F：GGTACCCGGGGATCCTCTAGAGTCGACAGAAGATGATATTGAAGG
		R：CTTTTGGTCTTCAGAAGAACTCGTCAAGAAGGC
pCAMBIA1300-h， pCAMBIA1300-heΔCcTLS1	CcITS-5	F：CCATGATTACGAATTCCTTAGATGTTCTGGGCCGCAC
		R：CTAGAGGATCCCCGGGTACCTACTTCCTCTAAATGACCGAGTTTG
	CcITS-3	F：AACAGCTCCCTCTAGAAAAGAAACCAACAGGGATTGCC
		R：GCAGGTCGACTCTAGAGGGAGCTGTTGGCTGGCTGG
	CcITS-h	F：ATTTAGAGGAAGTAGGTACCGGGGAGAGGCGGTTTGCG
		R：GCAGGTCGACTCTAGAGGGAGCTGTTGGCTGGCTGG
	CcTLS1-I	F：ATTTAGAGGAAGTAGGGCAGGTACTGGTTCGGATGT
		R：AACCGCCTCTCCCCGACAGCCTTTCGGCGGTTAGA
突变株验证 Mutant validation	FhphU	TGTCCTGCGGGTAAATAGC	Fu et al.，2012
	FhphL	TTGTTGGAGCCGAAATCC
	FrbU	CCTCTTCGCTATTACGCC
	FrbUC	GGCGTAATAGCGAAGAGG
	M13R	CAGGAAACAGCTATGACC	通用引物 Universal primer
	FhphLL	GGATTTCGGCTCCAACAA
	F5F	TTGTTGGAGCCGAAATCC
	F3F	AATTGCGATAACGAACGAGACC
	F4F2	GAATTAGCTGCGCTCATGCAC
	Neo-G418	F：GTCGACAGAAGATGATATTG	张晓珂，2015
		R：TCAGAAGAACTCGTCAAGAAGGCG	张晓珂，2015
	qSdhB	F：GGCGAAGTTGCCGTGAG
		R：CTTGGTGGTATCGGTGGG
	qCcTLS1	F：GACGAGGATGCTGATGAGGA
		R：TGTCGTACTGGGATGCTGTT

表2 基因相对表达量测定所用引物

Table 2 Primers for relative expression determination

基因 Gene	引物 Primer	序列（5′-3′） Sequence	参考文献 Reference
CcstuA	qstuA	F：GATCCATACCGCCCAGACAG
CcstuA		R：CGAGTACGCTGGCTTATCGT
CcHog1	qHog1	F：TGTCGACCCGATACTACCGT
		R：GCGTAATGTGTTCTCGCTGC
CcSCD1	qSCD1	F：TGTAATGGGCTGCCAGAGTG
		R：CCAGCTCTTGTCCAGGAAGG
CCK1	qCCK1	F：AAAGGACTACCACCACCAGC
		R：TCTTGGGGAACATGGCCTTC
Ccflbc	qflbc	F：GATCAGGTATTGCCCTCCCG
		R：TCGGGTGCTGTTGCCATATT
Cas2	qCas2	F：TCTGCTTTTGTAGCAGCCGT
		R：TAACAACCCGAACAAGCCCA
EF4α	qEF4α	F：TCACCGTCATTGACGCCC	Shi et al.，2021a
		R：CGGCAGCGATAATGAGGATAG	Shi et al.，2021a

图1 多主棒孢菌强致病力菌株HG14102524 CcTLS1敲除（A）和回补（B）菌株PCR及SdhB、CcTLS1（C、D）标准曲线 WT1：HG14102524野生型；P：敲除载体质粒；ΔC：敲除菌株；CP：回补载体质粒；cΔC：回补菌株。下同。

Fig. 1 PCR of virulent Corynespora cassiicola HG14102524 CcTLS1 knockout（A）and complementary（B）strains and standard curves of SdhB，CcTLS1（C，D） WT1：HG14102524 Wild-type；P：Knockout vector plasmid；ΔC：Knockout strain；CP：Complementary vector plasmid；cΔC：Complementary strain. The same below.

图2 多主棒孢菌弱致病力菌株HG15052104的CcTLS1异源表达菌株的PCR（A）及其CcTLS1表达的qRT-PCR（B） WT2：HG15052104野生型；P：异源表达载体质粒；AH3-23：异源表达菌株；ΔCcTLS1：敲除菌株。下同。

Fig. 2 PCR of low virulent Corynespora cassiicola HG15052104 CcTLS1 heterologous expression strains（A）and qRT-PCRof CcTLS1 expression（B） WT2：HG15052104 Wild-type：P：Heterologous expression vector plasmid；AH3-23：Heterologous expression strain；ΔCcTLS1：Knockout strain. The same below.

图3 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1突变菌株在活体黄瓜植株的致病力 H1-2：潮霉素表达菌株。下同。

Fig. 3 Pathogenicity of CcTLS1 mutants of Corynespora cassiicola virulent strain HG14102524 and low virulent strain HG15052104 in vivo cucumber plants H1-2：Hygromycin expressing strain. The same below.

图4 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1突变菌株在离体黄瓜叶片的致病力

Fig. 4 Pathogenicity of CcTLS1 mutants of Corynespora cassiicola virulent strain HG14102524 and low virulent strain HG15052104 in vitro cucumber plants

图5 多主棒孢菌强致病力菌株HG14102524分生孢子接种黄瓜叶片侵染过程显微形态观察 A ~ C：野生型菌株接种12 h；D ~ F：敲除菌株ΔCcTLS1接种12 h；G、H：野生型菌株接种24 h；I：敲除菌株ΔCcTLS1接种24 h。Co：分生孢子；Gt：芽管；Ap：类似附着孢结构；M：菌丝体；HR：过敏反应。

Fig. 5 Observation on micromorphology of cucumber leaves infected by conidia of Corynespora cassiicola virulent strain HG14102524 A-C：Inoculation with HG14102524 wild-type for 12 h；D-F：Inoculation with knockout strain ΔCcTLS1 for 12 h；G，H：Inoculation with HG14102524 Wild-type for 24 h；I：Inoculation with knockout strain ΔCcTLS1 for 24 h；Co：Conidia；Gt：Germ tube；Ap：Similar to appressorium structure；M：Mycelium；HR：Hypersensitive response.

图6 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1突变菌株在PDA上的菌落形态

Fig. 6 Colonial morphology of CcTLS1 mutant of Corynespora cassiicola virulent strain HG14102524 and low virulents train HG15052104 on PDA

图7 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1突变菌株菌落直径和产孢量在PDA培养基上生长5 d。* P < 0.05，** P < 0.01。下同。

Fig. 7 Colony diameter and conidiation of CcTLS1 mutant of Corynespora cassiicola virulent strain HG14102524 and low virulent strain HG15052104 on PDA On PDA medium for 5 days. * P < 0.05，** P < 0.01. The same below.

图8 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1突变菌株的菌丝形态特征

Fig. 8 Morphology of hyphae under microscope of CcTLS1 mutant of Corynespora cassiicola virulent strain HG14102524 and low virulent strain HG15052104 on PDA

图9 多主棒孢菌强致病力菌株HG14102524和弱致病力菌株HG15052104的CcTLS1的突变菌株纤维素酶分泌测定

Fig. 9 Determination of mutant to cellulase secretion of CcTLS1 mutant of Corynespora cassiicola virulent strain HG14102524 and low virulent strain HG15052104

图10 多主棒孢菌强致病力菌株HG14102524的CcTLS1的突变菌株中致病相关基因的相对表达量 A：产孢相关基因；B：蛋白激酶基因；C：黑色素合成关键基因。

Fig. 10 Relative expression levels of disease-related genes in CcTLS1 knockout mutants A：Sporulation-related genes；B：Protein kinase gene；C：Key genes for melanin synthesis.

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