Exploration on the Function of Pathogenicity-related Gene CcTLS1 in Corynespora cassiicola From Cucumber

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (3): 569-582.doi: 10.16420/j.issn.0513-353x.2021-1056

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Exploration on the Function of Pathogenicity-related Gene CcTLS1 in Corynespora cassiicola From Cucumber

WANG Quancheng, WU Jun, LI Lei, SHI Yanxia, XIE Xuewen, LI Baoju^*(), CHAI Ali^*()

Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2022-08-23 Revised:2022-10-19 Online:2023-03-25 Published:2023-04-03
Contact: *（E-mail：chaiali@caas.cn，libaojuivf@163.com）

Abstract

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

CLC Number:

S642.2

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.

Figures/Tables 12

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

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

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.

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.

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.

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

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.

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

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.

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

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

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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Exploration on the Function of Pathogenicity-related Gene CcTLS1 in Corynespora cassiicola From Cucumber

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