OsbHLH59 involved in rice resistance to Nilaparvata lugens(St?l) by regulating the expression level of xylanase inhibitor protein OsXIP

doi:10.3785/j.issn.1008-9209.2021.07.072

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

Issue (4): 453-464 DOI: 10.3785/j.issn.1008-9209.2021.07.072

Plant protection

OsbHLH59 involved in rice resistance to Nilaparvata lugens(St?l) by regulating the expression level of xylanase inhibitor protein OsXIP

Xueqi Lü¹(

),Ying XU¹,Yingying HUANG¹,Mingqi LIU²,Xiaoyan WENG¹(

)

^1.Institute of Plant Biology, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
^2.College of Life Sciences, China Jiliang University, Hangzhou 310018, China

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Abstract

Xylanase inhibitor protein (XIP) is considered to participate in plant defense. OsXIP was found to be a stress-responsive gene of Nilaparvata lugens (St?l) in the previous study. The transcription factor OsbHLH59 could be combined with the promoter of OsXIP in response to brown planthopper (BPH) treatment. In order to study whether OsbHLH59 was involved in regulating the expression of OsXIP and mediating rice resistance to BPH, OsbHLH59 overexpression transgenic lines were obtained by transgenic methods, and OsbHLH59 mutant lineswere obtained by using CRISPR/Cas9 system. The results showed that the expression levels of xylanase inhibitor OsXIP enhanced accompanied with the increased overexpression level of transcription factor gene OsbHLH59. Through the determination of agronomic traits, it was found that the growth of OsbHLH59 overexpression lines and OsXIP overexpression lines was less affected by the BPH treatment compared with wild type (WT). When WT plants and transgenic plants were exposed to BPH, the feeding preference of BPH in OsbHLH59 overexpression and OsXIP overexpression lines reduced compared with that of WT. With the BPH treatment, higher expression levels of defense-related genes were found in OsbHLH59 overexpression and OsXIP overexpression lines. In addition, compared with WT, OsbHLH59 overexpression and OsXIP overexpression lines had stronger abilities to remove excess H₂O₂ and higher antioxidant enzyme activities. This study reveals that OsbHLH59 can activate the expression of OsXIP, and OsXIP participates in rice resistance to BPH.

Key words： xylanase inhibitor protein OsXIP OsbHLH59 gene expression plant defence

Published: 25 August 2022

CLC:

Q 943.2

Fund: National Natural Science Foundation of China(30971702);Research Grants from the Science and Technology Department of Zhejiang Province, China(LGN19C130004)

Corresponding Authors: Xiaoyan WENG E-mail: 510557258@qq.com;xyweng@zju.edu.cn

About author: Lü Xueqi (https://orcid.org/0000-0003-0566-9494), E-mail: 510557258@qq.com

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Cite this article:

Xueqi Lü, Ying XU, Yingying HUANG, Mingqi LIU, Xiaoyan WENG. OsbHLH59 involved in rice resistance to Nilaparvata lugens(St?l) by regulating the expression level of xylanase inhibitor protein OsXIP. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(4): 453-464.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.07.072 OR https://www.zjujournals.com/agr/Y2022/V48/I4/453

转录因子OsbHLH59通过调控木聚糖酶抑制蛋白OsXIP表达水平影响水稻抗褐飞虱的机制研究（英文）

木聚糖酶抑制蛋白（xylanase inhibitor protein, XIP）在植物防御中发挥作用，实验室前期研究发现，水稻木聚糖酶抑制蛋白OsXIP能响应褐飞虱［Nilaparvata lugens (St?l)］胁迫。转录因子OsbHLH59可以与OsXIP的启动子结合以响应褐飞虱侵害。本研究利用转基因技术获得OsbHLH59过表达水稻株系并利用CRISPR/Cas9系统获得了OsbHLH59突变水稻株系，研究了转录因子OsbHLH59对OsXIP表达水平的影响及其在水稻抗褐飞虱中的作用。结果表明，OsbHLH59的过表达引起了OsXIP表达水平的上调。褐飞虱侵害对OsbHLH59过表达株系和OsXIP基因表达水平高的株系的生长水平影响相较野生型更小。与野生型相比，过表达OsbHLH59和OsXIP基因均降低了褐飞虱的取食偏好。同时，OsbHLH59过表达株系和OsXIP过表达株系的防御相关基因表达水平更高，H₂O₂含量更低，保护性酶活性也更高。综上所述，OsbHLH59可以提高OsXIP的表达，OsXIP在水稻响应褐飞虱胁迫中发挥了重要作用。

关键词： 木聚糖酶抑制蛋白, OsXIP, OsbHLH59, 基因表达, 植物防御

Table 1 Primers used for plasmid construction

Table 2 Primers used for qRT-PCR analysis

Fig. 1 Identification of OsbHLH59 mutant rice and relative expression levels of OsbHLH59 and OsXIP in overexpression lines, mutant lines and WT plantsA. Relative expression levels of OsbHLH59 in OsbHLH59 overexpression rice plants; B. Relative expression levels of OsbHLH59 in OsbHLH59 mutant rice plants; C. Relative expression levels of OsXIP in the six lines. Different lowercase letters above bars indicate significant differences at the 0.05 probability level. Data represent means ± standard deviation (SD) of three independent replicates. D. DNA level sequencing of OsbHLH59 mutants. The dashed lines indicate deleted nucleotides. Red uppercase letters indicate inserted or substituted nucleotides. The numbers on the right indicate the type of mutation and the number of nucleotides involved. i, d and s stand for nucleotide insertion, deletion and substitution, respectively.

Table 3 Net photosynthetic rates of transgenic plants at different stages (μmol/(m²·s))

Fig. 2 Growth characteristics of overexpression lines, mutant lines and WT after the BPH treatmentCompared with WT, single asterisk (*) and double asterisks (**) indicate significant and highly significant differences at the 0.05 and 0.01 probability levels, respectively.

Fig. 3 Overexpression of OsXIP and OsHLH59 positively regulating rice resistance to BPHA-E. Average number of BPH per plant on pairs of lines after exposure for 1-48 h. Data represent means ± SD of five independent replicates. Compared with WT, single asterisk (*) and double asterisks (**) indicate significant and highly significant differences at the same treatment time at the 0.05 and 0.01 probability levels, respectively. F. Damaged phenotypes of six rice lines infected by fourth-instar BPH alone.

Fig. 4 Relative expression of OsbHLH59 and OsXIP in the overexpression lines, mutant lines and WT after the BPH treatmentDifferent lowercase letters above bars indicate significant differences among different lines at the same treatment time at the 0.05 probability level, and the same as below.

Fig. 5 Relative expression levels of defense-related genes in the overexpression lines, mutant lines and WT after the BPH treatment

Fig. 6 Changes in H₂O₂ contents and antioxidant enzyme activities in overexpression lines, mutant lines and WT after the BPH treatment


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