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

摘要/Abstract

摘要：

【目的】通过分析发现木尔坦棉花曲叶病毒（cotton leaf curl Multan virus,CLCuMuV）C4开放阅读框（open reading frame,ORF）附近可编码3个不同大小的蛋白,而NCBI数据库登录的CLCuMuV各个分离物编码的C4蛋白大小也不尽相同,本研究试图通过分析该3个假定的“C4 ORF”编码蛋白对病毒致病性的影响,以明确CLCuMuV C4蛋白ORF的确切定位。【方法】根据双生病毒编码蛋白的保守性,在CLCuMuV C4 ORF附近查找到3个大小不等的ORF,分别标记为C4-L（567 nt）、C4-M（546 nt）和C4-S（303 nt）。利用同源重组的方法将该3个ORF分别构建到PVX异源表达载体,通过农杆菌介导的植物接种法,分析PVX介导的3个蛋白在本氏烟表达对本氏烟症状的影响。利用同源重组的方法构建CLCuMuV野生型及C4-L或C4-S缺失突变型的侵染性克隆,同样通过农杆菌介导的植物接种法,分别与其伴随DNA-β分子的侵染性克隆接种本氏烟,分析C4-L或C4-S缺失对CLCuMuV致病性的影响,并利用Southern blot和Western blot分别对接种烟草的病毒基因组的积累量和病毒C4蛋白的表达量进行分析。同时利用Gateway系统载体对C4-L和C4-S蛋白在本氏烟叶片表皮细胞中的亚细胞定位进行分析。【结果】PVX异源表达载体接种本氏烟,结果发现PVX-C4-L和PVX-C4-S接种的本氏烟叶片卷曲、叶柄伸长,并且PVX-C4-S症状更重,而PVX-C4-M接种的本氏烟症状较轻或基本无症状,Western blot试验也在PVX-C4-L和PVX-C4-S接种组检测到了C4蛋白的表达,说明C4-S ORF编码的蛋白对PVX异源病毒的致病性影响最大。侵染性克隆接种本氏烟,结果发现野生型（CLCuMuV）和C4-L突变型（CLCuMuV-ΔL）接种的本氏烟叶片皱缩增生、叶柄和茎秆扭曲,而C4-S突变型（CLCuMuV-ΔS）和对照组（Mock）接种的本氏烟未显示任何症状,并且前两者的植株高度明显矮于后两者,同时Southern blot试验在CLCuMuV和CLCuMuV-ΔL接种的本氏烟中均检测到大量病毒基因组的积累,而Western blot试验也在其中检测到C4蛋白的表达,说明C4-S ORF编码的蛋白在CLCuMuV诱导寄主产生症状过程中起关键作用。亚细胞定位发现YFP-C4-L主要定位在本氏烟叶片表皮细胞的叶绿体,YFP-C4-S则定位在细胞膜或细胞质周边,并有点状聚集体结构。【结论】C4-S ORF编码的蛋白对CLCuMuV的侵染必不可少,而C4-L和C4-M ORF编码的蛋白对CLCuMuV的侵染不是必需。

关键词: 木尔坦棉花曲叶病毒, C4开放阅读框, 致病性

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

郑信诗,尚鹏祥,李景远,丁新伦,吴祖建,张洁. 木尔坦棉花曲叶病毒“C4 ORF”编码蛋白对病毒致病性的影响[J]. 中国农业科学, 2021, 54(10): 2095-2104.

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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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.10.006

https://www.chinaagrisci.com/CN/Y2021/V54/I10/2095

图/表 5

表1

本研究中所用引物序列"

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

表1

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