木尔坦棉花曲叶病毒“C4 ORF”编码蛋白对病毒致病性的影响

doi:10.3864/j.issn.0578-1752.2021.10.006

Abstract

Abstract:

【Objective】Cotton leaf curl Multan virus (CLCuMuV) was found to encode three proteins with different sizes near the open reading frame (ORF) of C4, and the size of C4 protein encoded by each isolate of CLCuMuV registered in NCBI database was not the same. The objective of this study is to analyze the effect of proteins encoded by three hypothesized “C4 ORFs” on viral pathogenicity, and to determine the exact location of C4 ORF of CLCuMuV.【Method】According to the conserved protein of geminiviruses, three ORFs with different sizes were found near C4 ORF of CLCuMuV, which were labeled as C4-L (567 nt), C4-M (546 nt) and C4-S (303 nt). The three ORFs were constructed into PVX heterologous expression vector by homologous recombination, respectively. Using an agroinoculation method, the effect of PVX mediated expression of the three proteins on the symptoms of Nicotiana benthamiana were analyzed. Using a homologous recombination method, the infectious clones of CLCuMuV wild type and C4-L or C4-S deletion mutant were constructed, and then together with infectious clone of the associated DNA-β, they were inoculated into N. benthamiana respectively by agroinoculation. The effect of C4-L or C4-S deletion on CLCuMuV pathogenicity was analyzed. Meanwhile, Southern blot and Western blot were used to analyze the accumulation of viral genome and the expression level of viral C4 protein. At the same time, the subcellular localization of C4-L and C4-S proteins in the epidermal cells of N. benthamiana leaves was analyzed by a Gateway system vector.【Result】In the PVX heterologous expression assay, the results showed that the leaf curling and petiole elongation were observed in PVX-C4-L and PVX-C4-S inoculated N. benthamiana plants, and the symptoms of PVX-C4-S inoculated plants were more severe, while those of PVX-C4-M inoculated plants were mild or almost asymptomatic. The expression of C4 protein was also detected by Western blot in PVX-C4-L and PVX-C4-S inoculated plants. The results revealed that the protein encoded by C4-S ORF had the greatest effect on the pathogenicity of PVX heterologous virus. In the infectious clone assay, the results showed that the wild type (CLCuMuV) and C4-L mutant (CLCuMuV-ΔL) inoculated N. benthamiana plants had leaf wrinkled, petiole and stem distorted, while those inoculated with C4-S mutant (CLCuMuV-ΔS) and control (Mock) did not show any symptoms. Furthermore, the height of the CLCuMuV and CLCuMuV-ΔL inoculated plants was significantly shorter than that of CLCuMuV-ΔS and Mock inoculated plants. At the same time, the accumulation of viral genome was detected by Southern blot in both CLCuMuV and CLCuMuV-ΔL inoculated plants, and the expression of C4 protein was also detected by Western blot. The results revealed that the protein encoded by C4-S ORF played a key role in the process of CLCuMuV induced symptoms. The subcellular localization showed that YFP-C4-L was mainly located in the chloroplast of epidermal cells of N. benthamiana leaves, while YFP-C4-S was located in the cell membrane or the cytoplasmic periphery, and formed a point like aggregate structure. 【Conclusion】The protein encoded by C4-S ORF is essential for the infection of CLCuMuV, while the proteins encoded by C4-L and C4-M ORF are not necessary.

Key words: cotton leaf curl Multan virus (CLCuMuV), C4 open reading frame (C4 ORF), pathogenicity

ZHENG XinShi,SHANG PengXiang,LI JingYuan,DING XinLun,WU ZuJian,ZHANG Jie. Effects of Proteins Encoded by “C4 ORFs” of Cotton Leaf Curl Multan Virus on Viral Pathogenicity[J].Scientia Agricultura Sinica, 2021, 54(10): 2095-2104.

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Figures/Tables 5

Table 1

Primers used in this study"

引物用途及名称 Application and name of primer	引物序列^a Primer sequence (5′- 3′)	引物在病毒基因组中的位置^b Position of primers in viral genome
PVX异源表达For PVX heterologous expression
C4-L-F	cagctagcatcgattcccgggATGATTTCTCAAATTACGATAATGC	2694-2670
C4-M-F	cagctagcatcgattcccgggATGCCATTTGGAGACACTAT	2673-2654
C4-S-F	cagctagcatcgattcccgggATGGGAGCCCTCATCTCCAT	2430-2411
C4-R	cttatcggcggtcgacccgggCTAGTTCCTTAATGACTCTAAGAGC	2128-2152
PVX-F	CCCTATCAAGCTTATCGGCGGTC	--
PVX-R	CATTGCCGATCTCAAGCCACTCTC	--
亚细胞定位For subcellular localization
C4-L-221-F	ggggacaagtttgtacaaaaaagcaggcttcATGATTTCTCAAATTACGATAATG	2694-2671
C4-221-R	ggggaccactttgtacaagaaagctgggtcGTTCCTTAATGACTCTAAGAGCC	2131-2153
M13-F	TGTAAAACGACGGCCAGT	--
M13-R	CAGGAAACAGCTATGACC	--
Gateway-F	GGGCTGGCAAGCCACGTTTGGTG	--
Gateway-R	CCGGGAGCTGCATGTGTCAGAGG	--
侵染性克隆For infectious clones
CLCuMuV-L-F	acgccaagctggcgcgccaagcttGATCCATTGTTAAACGAATTTCCTGATACG	123-152
CLCuMuV-L-R	TTTAACAATGGATCCCACATgtttgaatttgaaacttagtgcgca	136-117
CLCuMuV-R-F	ATGTGGGATCCATTGTTAAACGAATTTCC	117-145
CLCuMuV-R-R	agaattcgagctcggtacccgggcacatGTTTGAATTTGAAACTTAGTGCGCA	116-92
pBIN-F	GTATGTTGTGTGGAATTGTGAGCGGATAAC	--
pBIN-R	CACTGGCCGTCGTTTTACAACGTCGTGAC	--
C4L-i2o-R-pBIN-F	TCACATTTCAAAATCCTCACGCTCCAAAAAG	2692-2719
C4L-i2o-L-pBIN-R	GGAGCGTGAGGATTTTGAAATGTGATCTCAAATTACGATAATGCCATTTG	2713-2692, 2688-2664
C4S-G2O-R-pBIN-F	ACATCTTCGTGTAACTCTCTGCAGAC	2430-2452
C4S-G2O-L-pBIN-R	TCTGCAGAGAGTTACACGAAGATGTGAGCCCTCATCTCCATGTGCTCATC	2451-2428, 2424-2402
CLCuMuV-RBamH1-pBIN-F	TTCAAATTCAAACATGTGGGATCCATTGTTAAACGAATTTCCTG	104-147
CLCuMuV-CPprobe-F	GCAGCCCATACACCAGCCGTGTTGC	347-371
CLCuMuV-CPprobe-R	GTTCCTTACTCGCATATTGACCACC	862-838
CLCuMuV-BETAprobe-F	GTCCCACTGCTGGTATTGACTTGATTTG	37-64
CLCuMuV-BETAprobe-R	CCATGAAATAAGCAGATATGACGAGGAGC	567-539

Table 1

Fig. 1

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Fig. 4

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Effects of Proteins Encoded by “C4 ORFs” of Cotton Leaf Curl Multan Virus on Viral Pathogenicity

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