Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

doi:10.1016/S2095-3119(14)60937-0

Journal of Integrative Agriculture ›› 2015, Vol. 14 ›› Issue (9): 1767-1777.DOI: 10.1016/S2095-3119(14)60937-0

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

CHENG Chun-zhen, YANG Jia-wei, YAN Hu-bin, BEI Xue-jun, ZHANG Yong-yan, LU Zhi-ming, ZHONG Guang-yan

1、College of Horticulture and landscape architecture, Southwest University, Chongqing 400715, P.R.China
2、Institute of Fruit Tree Research, Guangdong academy of agricultural Sciences, Guangzhou 510640, P.R.China
3、Economic Crops Station, agricultural Bureau of Rongxian, Zigong 643100, P.R.China
4、Institute of Crop Science, Shanxi academy of agricultural Sciences, Taiyuan 030031, P.R.China
5、Key laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of agriculture, Guangzhou 510640,P.R.China

收稿日期:2014-09-25 出版日期:2015-09-03 发布日期:2015-09-05
通讯作者: ZHONG Guang-yan, E-mail: gy_zhong@163.com
作者简介:CHENG Chun-zhen, E-mail: ld0532cheng@126.com; YANG Jia-wei,E-mail: yangjiawei1010@163.com; YAN Hu-bin, E-mail: maikyhb@ 126.com; * These authors contributed equally to this study.
基金资助:
This work was supported by the International Science & Technology Cooperation Program of China (2012DFA30610), the National Natural Science Foundation of China (30571291) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201203075-07).

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

CHENG Chun-zhen, YANG Jia-wei, YAN Hu-bin, BEI Xue-jun, ZHANG Yong-yan, LU Zhi-ming, ZHONG Guang-yan

1、College of Horticulture and landscape architecture, Southwest University, Chongqing 400715, P.R.China
2、Institute of Fruit Tree Research, Guangdong academy of agricultural Sciences, Guangzhou 510640, P.R.China
3、Economic Crops Station, agricultural Bureau of Rongxian, Zigong 643100, P.R.China
4、Institute of Crop Science, Shanxi academy of agricultural Sciences, Taiyuan 030031, P.R.China
5、Key laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of agriculture, Guangzhou 510640,P.R.China

Received:2014-09-25 Online:2015-09-03 Published:2015-09-05
Contact: ZHONG Guang-yan, E-mail: gy_zhong@163.com
About author:CHENG Chun-zhen, E-mail: ld0532cheng@126.com; YANG Jia-wei,E-mail: yangjiawei1010@163.com; YAN Hu-bin, E-mail: maikyhb@ 126.com; * These authors contributed equally to this study.
Supported by:
This work was supported by the International Science & Technology Cooperation Program of China (2012DFA30610), the National Natural Science Foundation of China (30571291) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201203075-07).

摘要/Abstract

摘要： The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strategies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA fragments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNA was sufficient to confer sour orange with CTV resistance/tolerance.

关键词: Citrus tristeza virus (CTV) , RNA interference , transgenic plant , Citrus aurantium , disease resistance

Abstract: The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strategies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA fragments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNA was sufficient to confer sour orange with CTV resistance/tolerance.

Key words: Citrus tristeza virus (CTV) , RNA interference , transgenic plant , Citrus aurantium , disease resistance

CHENG Chun-zhen, YANG Jia-wei, YAN Hu-bin, BEI Xue-jun, ZHANG Yong-yan, LU Zhi-ming, ZHONG Guang-yan. Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains[J]. Journal of Integrative Agriculture, 2015, 14(9): 1767-1777.

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Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

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