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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2022, Vol. 48 Issue (6): 753-760    DOI: 10.3785/j.issn.1008-9209.2022.07.111
Research articles     
Molecular mechanisms underlying the regulation of tobacco defenses against Bemisia tabaci by the salivary effector BtArmet targeting NtWRKY51
Hui DU(),Xiaowei WANG,Shusheng LIU()
Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
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Abstract  

The whitefly Bemisia tabaci, an important agricultural insect pest, secretes saliva into plants while feeding on them. Our previous study found that the whitefly secretes the salivary effector protein BtArmet (Bemisia tabaci arginine rich, mutated in early stage of tumors) to inhibit plant resistance by targeting tobacco cystatin. In this study, we continued to screen tobacco proteins that interact with the salivary effector protein BtArmet. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that Nicotiana tabacum WRKY51 interacted with BtArmet. The up-regulation of NtWRKY51 gene in tobacco was significantly induced by whitefly infestation. However, virus-induced gene silencing (VIGS) assays combined with bioassay of whiteflies showed that the fecundity of NtWRKY51-silenced tobacco plants by female whiteflies was significantly lower than that of the negative control (tobacco with silenced empty vector). Silencing NtWRKY51 gene had no effect on phytohormone signaling pathways mediated by salicylic acid or jasmonic acid in tobacco plants. These results provide references for further investigations on the molecular mechanisms underlying plant resistance to whiteflies.

Key words： whitefly      saliva      tobacco      plant defense      WRKY51      insect-plant interaction     
Received: 11 July 2022      Published: 27 December 2022
CLC:  S 436  
Corresponding Authors: Shusheng LIU     E-mail: duhui8023@163.com;shshliu@zju.edu.cn
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Hui DU
Xiaowei WANG
Shusheng LIU
Cite this article:

Hui DU,Xiaowei WANG,Shusheng LIU. Molecular mechanisms underlying the regulation of tobacco defenses against Bemisia tabaci by the salivary effector BtArmet targeting NtWRKY51. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(6): 753-760.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.07.111     OR     https://www.zjujournals.com/agr/Y2022/V48/I6/753


唾液效应因子BtArmet靶向NtWRKY51调控烟草防御烟粉虱的分子机制

重要农业害虫烟粉虱（Bemisia tabaci）具刺吸式口器，在取食植物韧皮部汁液的同时将唾液分泌到植物中。前期研究发现，烟粉虱通过分泌唾液效应蛋白BtArmet（Bemisia tabaci arginine rich, mutated in early stage of tumors）靶向烟草体内的半胱氨酸蛋白酶抑制素蛋白，抑制植物的抗虫性。本研究在前期研究的基础上继续筛选与烟粉虱唾液效应蛋白BtArmet互作的烟草蛋白。通过酵母双杂交和双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验发现，普通烟的转录因子WRKY51可以与BtArmet发生互作。烟粉虱的侵染可以显著诱导烟草中NtWRKY51基因表达上调，但病毒诱导的基因沉默（virus-induced gene silencing, VIGS）方法结合烟粉虱生物学测定实验显示，烟粉虱在沉默NtWRKY51基因烟草上的产卵量显著低于阴性对照（沉默空载的烟草），且沉默NtWRKY51基因烟草中水杨酸和茉莉酸介导的激素信号通路均不受影响。上述结果为进一步探究植物对烟粉虱的抗性及其分子机制奠定了基础。


关键词： 烟粉虱,  唾液,  烟草,  植物防御,  WRKY51,  昆虫-植物互作 

引物名称

Primer name

基因名称/用途

Gene name/usage

引物序列（5′→3′）

Primer sequence (5′→3′)

BtArmet-BKBtArmet/Y2HF: GGAATTCATGGCAACACCAAGCGAT
R: CGGGATCCTTACAATTCATCGCGGACAT
BtArmet-p2YCBtArmet/BiFCF: CCCTTAATTAACATGGCAACACCAAGCGATG
R: GGGACTAGTCAATTCATCGCGGACATAT
NtWRKY51-p2YNNtWRKY51/BiFCF: CCCTTAATTAACATGAACTCCACCTTCCTAG
R: GGGACTAGTAACACCATTGGGAATTGCTT
NtWRKY51 qRTNtWRKY51/qRT-PCRF: TGGAGGAAGTATGGGAAGAAGA
R: GCTTTCATGGTTATGTCTGCCT
NtWRKY51 VIGSNtWRKY51/VIGSF: CGGGATCCATGAACTCCACCTTCCTAG
R: GCTCTAGAATTCATCTTTTCTTTCTTTATCC
qPALPAL/qRT-PCRF: AAGAAGCGTTCCGTGTTGCTG
R: TCGGGCTTTCCATTCATCACC
qICSICS/qRT-PCRF: CAGTTCTGTTTGCAACCTCC
R: CGCTGGAACATACAGCCTC
qBGL2BGL2/qRT-PCRF: TAGGAAACAACATCAAGGTCTCAAC
R: TCCCTACAGATGCCCCTCC
qFAD7FAD7/qRT-PCRF: CATGTGGCTTGACTTAGTTACCTACT
R: CCCTGACTTCTTTGGCTCCTT
qCOI1COI1/qRT-PCRF: GAGAGGAGAGAATAACAGATC
R: AGCAGCCTCTCACTTCTAGC
qPI2PI2/qRT-PCRF: ACTACTTCGGTCAGGATGG
R: AACGGGCAACTTATGGTA
qGAPDHGAPDH/qRT-PCRF: GCAGTGAACGACCCATTTATCTC
R: AACCTTCTTGGCACCACCCT
Table 1 Primers used for gene cloning, vector construction and quantitative detection
Fig. 1 Sequence analysis of NtWRKY51Single asterisk (*) indicates the termination codon.
Fig. 2 Interactions of BtArmet and NtWRKY51 in the yeast two-hybrid systemBD: pGBKT7 empty vector; AD: pGADT7 empty vector; QDO: Quadruple dropout substrate; X: Chromogenic substrate of X-α-gal; A: Aureobasidin A; DDO: Double dropout substrate.
Fig. 3 Interactions of BtArmet and NtWRKY51 in vivoRFP: Red fluorescent protein; YFP: Yellow fluorescent protein. RFP was used as a marker of the nucleus. Unfused cYFP and nYFP proteins were used as negative controls.
Fig. 4 Relative expression level of NtWRKY51 in N. tabacum fed by whitefliesSingle asterisk (*) indicates significant differences at the 0.05 probability level, and n=4. The same as Fig. 6.
Fig. 5 Phenotype of NtWRKY51-silenced N. tabacumNegative control: Tobacco with silenced empty vector, and the same as below.
Fig. 6 Relative expression level of NtWRKY51 in N. tabacum
Fig. 7 Fecundity and survival rate of whiteflies on NtWRKY51-silenced N. tabacumDouble asterisks (**) indicate highly significant differences at the 0.01 probability level, and ns indicates no significant differences; n=20.
Fig. 8 Relative expression levels of marker genes in SA-signaling pathways and JA-signaling pathways in NtWRKY51-silenced N. tabacumThe symbol ns indicates no significant differences, and n=6.
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