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| 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 |
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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.
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Received: 04 January 2021
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Corresponding Authors:
* ldq@hebau.edu.cn; ndwanghaiyan@163.com
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[1] 崔钟池, 武文月, 王婉晴, 等. 2020. AvrLr19突变菌株诱导TcLr19小麦中差异表达基因分析[J]. 河北农业大学学报, 43(05): 1-7.
(Cui Z C, Wu W Y, Wang W Q, et al.2020. Analysis of differentially expressed genes in TcLr19 wheat induced by AvrLr19 mutant strains[J]. Journal of Hebei Agricultural University, 43(05): 1-7.)
[2] 范春霞, 王军节, 赵鲁迺克, 等. 2020. 甜瓜粉霉病菌效应蛋白编码基因的预测与分析[J]. 植物病理学报, 50(05): 549-560.
(Fan C X, Wang J J, Zhao L N K, et al.2020. Genome-wide prediction and analysis of candidate effector-encoding genes in Trochothecium roseum[J]. Acta Phytopathologica Sinica, 50(05): 549-560.)
[3] Abramovitch R B, Kim Y J, Chen S R, et al.2003. Pseudomonas TypeⅢ effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death[J]. The EBMO Journal, 22(1): 60-69.
[4] Almagro Armenteros J J, Tsirigos K D, Sonderby C K, et al.2019. SignalP 5.0 improves signal peptide predictions using deep neural networks[J]. Nature Biotechnology, 37(4): 420-423.
[5] Anderson C, Khan M A, Catanzariti A M, et al.2016. Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini[J]. BMC Genomics, 17(1): 667-687.
[6] Baek D, Name J, Lee J, et al.2004. BAX-induced cell death of Arabidopsisis meditated through reactive oxygen-dependent and-independent processes[J]. Plant Molecular Biology, 56(1): 15-27.
[7] Chen J, Upadhyaya N M, Ortiz D, et al.2017. Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat[J]. Science, 358(6370): 1607-1610.
[8] Emanuelsson O, Nielsen H, Brunak S, et al.2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[J]. Journal of Molecular Biology, 300(4): 1005-1016.
[9] Finn R D, Bateman A, Clements J, et al.2014. Pfam: The protein families database[J]. Nucleic Acids Research, 42: D222-D230.
[10] Jones J D, Dangl J L.2006. The plant immune system[J]. Nature, 444(7117): 323-329.
[11] Kemen E, Kemen A C, Rafiqi M, et al.2005. Identification of a protein from rust fungi transferred from haustoria into infected plant cells[J]. Molecular Plant-Microbe Interactions, 18(11): 1130-1139.
[12] Krogh A, Larsson B, von Heijne G, et al.2001. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes[J]. Journal of Molecular Biology, 305(3): 567-580.
[13] Ma L, Lukasik E, Gawehns F, et al.2012. The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves[J]. Methods in Molecular Biology, 835: 61-74.
[14] Petre B, Joly D L, Duplessis S.2014. Effector proteins of rust fungi edictor[J]. BMC Bioinformatics, 9: 392-403.
[15] Pierleoni A, Martelli P L, Casadio R.2008. PredGPI: A GPI-anchor predictor[J]. BMC Bioinformatics, 9(1): 1-11.
[16] Qi T, Guo J, Liu P, et al.2019. Stripe rust effector PstGSRE1 disrupts nuclear localization of ROS-promoting transcription factor TaLOL2 to defeat ROS-induced defense in wheat[J]. Molecular Plant, 12(12): 1624-1638.
[17] Salcedo A, Rutter W, Wang S, et al.2017. Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99[J]. Science, 358(6370): 1604-1606.
[18] Segovia V, Bruce M, Rupp J L, et al.2016. Two small secreted proteins from induce reduction of ß-glucoronidase transient expression in wheat isolines containing and Lr9, Lr24 and Lr26[J]. Canadian Journal of Plant Pathology, 38(1): 91-102.
[19] Sperschneider J, Dodds P N, Gardiner D M, et al.2018. Improved prediction of fungal effector proteins from secretomes with EffectorP 2.0[J]. Molecular Plant Pathology, 19(9): 2094-2110.
[20] Wei J, Cui L P, Zhang N, et al.2020. Puccinia triticina pathotypes THTT and THTS display complex transcript profiles on wheat cultivar Thatcher[J]. BMC genetics, 21(1): 48-59.
[21] Wu J Q, Sakthikumar S, Dong C, et al.2017. Comparative genomics integrated with association analysis identifies candidate effector genes corresponding to Lr20 in phenotype-paired Puccinia triticina isolates from Australia[J]. Frontiers Plant Science, 8: 148-166.
[22] Zhang Y, Wei J, Qi Y, et al.2020. Predicating the effector proteins secreted by Puccinia triticina through transcriptomic analysis and multiple prediction approaches[J]. Frontiers in Microbiology, 11: 538032.
[23] Zhao S Q, Shang X F, Bi W S, et al.2020. Genome-wide identification of effector candidates with conserved motifs from the wheat leaf rust fungus Puccinia triticina[J]. Frontiers Microbiology, 11: 1188-1203.
[24] Yang Q, Huai B Y, Lu Y X, et al.2020. A stripe rust effector Pst18363 targets and stabilises TaNUDX23 that promotes stripe rust disease[J]. New Phytologist, 225: 880-895. |
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