Screening and Analysis of Effector Candidates in Physiological Race PHNT of Puccinia triticina on Wheat (Triticum aestivum)
WU Wen-Yue, SUN Xin-Kang, QIN Zhen, MA Qiu-Ying, HAN Xi, WANG Ya-Min, LIU Da-Qun*, WANG Hai-Yan*
College of Plant Protection, Hebei Agricultural University/Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province, Baoding 071001, China
Abstract：The leaf rust (Puccinia triticina) in Triticum aestivum secretes various special effectors, which invade host cells and play a toxic role to destroy the plant defense response. It is of great significance to analyze the pathogenic mechanism of leaf rust for the development of disease control technologies and strategies. In previous study, leaf rust physiological race PHNT were mutagenized with ethyl methanesulfonate (EMS) and virulent mutants of leaf rust resistance gene Lr19 were isolated successfully. Then RNA sequencing was performed on samples collected from Lr19-virulent mutants and wild-type strains. In this study, Signal P, TMHMM, Big-PI Predictor, Target P and Effector P online prediction software was used to predict effector candidates. The results showed that a total of 301 effector candidates were screened out of 10 870 protein sequences of leaf rust, and the length of signal peptides ranged from 19 to 23 aa. The amino acids with the highest appearance frequency in signal peptides were leucine and alanine. The prediction results of subcellular localization showed that effector candidates were mainly located extracellular. Three new motifs were obtained by MEME prediction analysis. In order to determine the expression pattern of effector candidates in different time course of wheat infected by leaf rust, 7 candidate effector proteins rich in cysteine, glycine or serine were randomly selected from 301 candidate effector proteins, and their gene expression was analyzed by qRT-PCR. The results showed that 7 genes were up-regulated in the early stage of leaf rust infection, which might play a role in the process of leaf rust infection. In order to verify the toxic function of the effectors, 7 genes were constructed to pEarleyGate103 with GFP label by Gateway cloning. The toxicity function of effector candidates was verified by Agrobacterium tumefaciens-mediated transient transformation. The results showed that PTTG_27401 could inhibit BCL2-associated X (BAX)-induced cell necrosis which meant potential toxic function. The present study provides basic information for further identification of PHNT effectors and analysis of pathogenic mechanism.
武文月, 孙新康, 秦臻, 马秋颖, 韩茜, 王亚敏, 刘大群, 王海燕. 小麦叶锈菌生理小种PHNT候选效应蛋白的筛选及分析[J]. 农业生物技术学报, 2021, 29(8): 1463-1472.
WU Wen-Yue, SUN Xin-Kang, QIN Zhen, MA Qiu-Ying, HAN Xi, WANG Ya-Min, LIU Da-Qun, WANG Hai-Yan. Screening and Analysis of Effector Candidates in Physiological Race PHNT of Puccinia triticina on Wheat (Triticum aestivum). 农业生物技术学报, 2021, 29(8): 1463-1472.
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