Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (12): 2371-2384.doi: 10.3864/j.issn.0578-1752.2020.12.006

• PLANT PROTECTION • Previous Articles     Next Articles

Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31

QI Yue1,LÜ JunYuan1,ZHANG Yue1,WEI Jie1,ZHANG Na1,YANG WenXiang1(),LIU DaQun2()   

  1. 1 Wheat Leaf Rust Research Center, College of Plant Protection, Agricultural University of Hebei/Technological Innovation Center for Biological Control of Plant Diseases and Insect Pests of Hebei Province/National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071001, Hebei;
    2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-12-26 Online:2020-06-16 Published:2020-06-25
  • Contact: WenXiang YANG,DaQun LIU E-mail:wenxiangyang2003@163.com;liudaqun@caas.cn

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Abstract:

【Objective】 Wheat leaf rust caused by Puccinia triticina (Pt) is one of the main diseases affecting wheat production. In the process of interaction between wheat and Pt, the Pt will secrete some effector proteins to the host cells to regulate the host and play a virulent function. The objective of this study is to carry out the research on Pt effector proteins, explore the pathogenic mechanism of Pt, and to provide a basis for the continuous prevention and control of diseases. 【Method】 The cDNA of Pt 13-5-72 interacting with susceptible variety Thatcher was used as template to amplify effector protein Pt18906. Sequence features of Pt18906 were analyzed using SignalP 4.1, TargetP 1.1, TMHMM 2.0 and EffectorP 2.0 software. The online software Swiss-Model was used to predict the tertiary structure of Pt18906. The secondary structure of Pt18906 was predicted by online software SOPMA. The expression pattern of Pt18906 was analyzed by RT-qPCR. The verification that Pt18906 inhibited Bax and INF1-induced programmed cell death (PCD) was conducted using Nicotiana benthamiana heterogeneous expression system. The secretion function of Pt18906 signal peptide was verified by the yeast system. The deletion mutation of Pt18906 was used to identify the virulence function motif. Subcellular localization of Pt18906 was analyzed using transient expression of Pt18906-GFP in N. benthamiana cells. Transient expression technology was used to carry out the avirulent function of Pt18906 on a full set of near-isogenic lines that did not contain disease resistance genes and contained different disease resistance genes with Thatcher as the background. The transient transformation of wheat mediated by the TTSS (Type III secretion system) was used to analyze the activity of regulating the host defense response of Pt18906. 【Result】 A Pt effector Pt18906, significantly upregulated during the interaction between Pt and Thatcher, was obtained from the transcriptome library of interaction between Pt and Thatcher. The full-length ORF sequence with 672 bp was amplified in the cDNA of wheat leaves inoculated by Pt strain 13-5-72. The effector protein lacks known functional domains and motifs. Its working environment is alkaline. Pt18906 could inhibit the PCD induced by Bax from mice and INF1 from Oomycetes by transient expression in N. benthamiana cells, which indicates that the effector protein has virulent function. By constructing deletion mutants, it is clear that the 28-47 amino acids have an important role in its virulent function. The effector protein is located in the nucleus and cytoplasm, which indicates that it acts within the cells. Pt18906 could cause allergic necrosis in the disease-resistant varieties of single gene lines TcLr27+31 and TcLr42, indicating that the effector protein is avirulent. Pt18906 could cause the accumulation of reactive oxygen species (ROS) and callose in TcLr27+31 through transiently expressing using a bacterial type III secretion system. The callose increased gradually with the injection time, and ROS reached the highest level at 10 min after injection. 【Conclusion】 The main functional domain of Pt18906 is 28-47 amino acids. Pt18906 can trigger the two-layer defense reaction accompanied with the accumulation of callose in wheat TcLr27+31 and oxidative burst.

Key words: Puccinia triticina (Pt), wheat leaf rust, effector protein, callose, reactive oxygen species (ROS), pathogenicity, defense reaction

Table 1

The primers used in this study"

引物类型
Primer type		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 序列长度
Sequence length (bp)
Pt18906的ORF引物
Primer for Pt18906 ORF
信号肽分泌功能验证
Function verification of signal peptide secretion
异源瞬时表达
Heterogeneous transient expression



亚细胞定位
Subcellular localization
毒性结构域验证
Verification of virulent domain



细菌三型分泌系统
Bacterial type Ⅲ secretion system		 ORFPt18906-F
ORFPt18906-R
SPPt18906-F
SPPt18906-R
Pt18906-F
ΔSPPt18906-F
Pt18906-R
LBA
LBB
DPt18906-F
DPt18906-R
qPt18906-F1
qPt18906-R1
qPt18906-R2
qPt18906-R3
qPt18906-R4
PPt18906-F
PPt18906-R		 ATGTTTTCAGCAAGTTCAAT
CTACTTACCCTTCTCCTTAG
CCGGAATTCATGTTTTCAGCAAGTTCAAT
CCGCTCGAGAAGCTCAACGGGTGGTAAGC
TCCCCCGGGATGTTTTCAGCAAGTTCAAT
TCCCCCGGGGCCGAAGTCCAACGACACGC
GCGTCGACCTACTTACCCTTCTCCTTAG
CAATCACAGTGTTGGCTTGC
GACCCTATGGGCTGTGTTG
CCCATCGATGCCGAAGTCCAACGACACGC
TCCCCCGGGCTTACCCTTCTCCTTAGGAT
CCCATCGATGCTCCACTCAAAAACGGTGA
TCCCCCGGGTTGGTTGTTAACGTCCTCAA
TCCCCCGGGGACCGCCATGTATCCGGCTA
TCCCCCGGGGGGGTCTATCTCTGTAGGAT
TCCCCCGGGTATGCTCCCGTATTTGCATG
CACCATGTTTTCAGCAAGTTCAAT
CTACTTACCCTTCTCCTTAG		 20
20
29
29
29
29
28
21
20
29
29
29
29
29
29
29
24
20

Fig. 1

Sequence analysis of Pt18906"

Fig. 2

Pt18906 inhibits PCD induced by Bax and INF1 Verify that Pt18906 inhibits Bax and INF1-induced PCD (1: The inhibition of Bax-induced PCD 24 h after Pt18906 injection; 2: The inhibition of Bax-induced PCD at 0 h after Pt18906 injection; 3: The inhibition of PCD by INF1 24 h after Pt18906 injection; 4: The effective inhibition of PCD by INF1 0 h after Pt18906 injection)"

Fig. 3

Verification of signal peptide secretory function of Pt18906"

Fig. 4

Pt18906 expression at different time points in P. triticina 13-5-72 and Thatcher interaction"

Fig. 5

Pt18906 virulence functional domain analysis"

Fig. 6

Subcellular localization of Pt18906 in N. benthamiana"

Fig. 7

Overexpression of Pt18906 in wheat stimulates HR"

Fig. 8

Expression of Pt18906 in TcLr27+31 causes accumulation of callose"

Fig. 9

Expression of Pt18906 in TcLr27+31 causes ROS bursts"

[1] RATTU A R, AHMAD I, FAYYAZ M, AKHTAR M A, IRFAN-UL-HAQUE , ZAKRIA M, AFZAL S N. Virulence analysis of Puccinia triticinia cause of leaf rust of wheat. Pakistan Journal of Botany, 2009,41(4):1957-1964.
[2] ROELFS A P, SINGH R P, SAARI E E. Rust Disease of Wheat: Concepts and Methods of Disease Management. Mexico, DF: CMMYT, 1992: 7-14.
[3] KOLMER J A. Genetics of resistance to wheat leaf rust. Annual Review of Phytopathology, 1996,34:435-455.
[4] RAMACHANDRAN S R, YIN C T, KUD J, TANAKA K, MAHONEY A K, XIAO F M, HULBERT S H. Effectors from wheat rust fungi suppress multiple plant defense responses. Phytopathology, 2017,107(1):75-83.
pmid: 27503371
[5] 刘冰玉, 蔡宝珊, 高成江. 泛素化调控抗病毒天然免疫的研究进展. 中国科学: 生命科学, 2018,48(11):1152-1161.
LIU B Y, CAI B S, GAO C J. Regulation of innate antiviral immunity by protein ubiquitination. Scientia Sinica Vitae, 2018,48(11):1152-1161. (in Chinese)
[6] SCHORNACK S, BALLVORA A, GURLEBECK D, PEART J, GANAL M, BAKER B, BONAS U, LAHAYE T. The tomato resistance protein Bs4 is a predicted non-nuclear TIR-NB-LRR protein that mediates defense responses to severely truncated derivatives of AvrBs4 and overexpressed AvrBs3. The Plant Journal, 2004,37(1):46-60.
doi: 10.1046/j.1365-313x.2003.01937.x pmid: 14675431
[7] THOMMA B P H J, NUMBERGER T, JOOSTEN M H A J. Of PAMPs and effectors: The Blurred PTI-ETI dichotomy. The Plant Cell, 2011,23(1):4-15.
pmid: 21278123
[8] 李伟兰, 戎伟, 何朝族. 一个推测的野油菜黄单胞菌Ⅲ型分泌效应子效应子基因XopXccP对寄主植物的致病性分析. 植物病理学报, 2014,44(2):173-179.
LI W L, RONG W, HE C Z. XopXccP, a putative type Ⅲ effector gene of Xanthomonas campestris pv. campestris, is required for pathogenicity on host plants. Acta Phytopathologica Sinica, 2014,44(2):173-179. (in Chinese)
[9] CHASTAGNER P, ISRAEL A, BROU C. AIP4/Itch regulates notch receptor degradation in the absence of ligand. PLoS ONE, 2008,3(7):e2735.
pmid: 18628966
[10] ONG L E, INNES R W. AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis. Molecular Microbiology, 2006,60(4):951-962.
doi: 10.1111/j.1365-2958.2006.05162.x pmid: 16677306
[11] LI L, LI M, YU L, ZHOU Z, LIANG X, LIU Z, CAI G, GAO L, ZHANG X, WANG Y, CHEN S, ZHOU J M. The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host and Microbe, 2014,15(3):329-338.
[12] LU D, WU S, GAO X, ZHANG Y, SHAN L, HE P. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proceedings of the National Academy of Sciences of the United States of America, 2010,107(1):496-501.
doi: 10.1073/pnas.0909705107 pmid: 20018686
[13] MONAGHAN J, MATSCHI S, SHORINOLA O, ROVENICH H, MATEI A, SEGONZAC C, MALINOVSKY F G, RATHJEN J P, MACLEAN D, ROMEIS T, ZIPFEL C. The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover. Cell Host and Microbe, 2014,16(5):605-615.
doi: 10.1016/j.chom.2014.10.007 pmid: 25525792
[14] ZHANG J, LI W, XIANG T, LIU Z, LALUK K, DING X, ZOU Y, GAO M, ZHANG X, CHEN S, MENGISTE T, ZHANG Y, ZHOU J M. Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host and Microbe, 2010,7(4):290-301.
[15] 白志英, 王冬梅, 侯春燕, 刘娜, 韩胜芬, 马利华. 小麦叶锈菌侵染过程的显微和超微结构. 细胞生物学杂志, 2003,25(6):393-397.
BAI Z Y, WANG D M, HOU C Y, LIU N, HAN S F, MA L H. Microstructure and ultrastructure infected by wheat rust fungus. Chinese Journal of Cell Biology, 2003,25(6):393-397. (in Chinese)
[16] SPERSCHNEIDER J, GARDINER D M, DODDS P N, TINI F, COVARELLI L, SINGH K B, MANNERS J M, TAYLOR J M. EffectorP: Predicting fungal effector proteins from secretomes using machine learning. New Phytologist, 2016,210(2):743-761.
doi: 10.1111/nph.13794 pmid: 26680733
[17] TAKKEN F L, GOVERSE A. How to build a pathogen detector: Structural basis of NB-LRR function. Current Opinion in Plant Biology, 2012,15(4):375-384.
pmid: 22658703
[18] JAROSE A M, BURDON J J. Host-pathogen interactions in natural populations of Linum marginale and Melampsora lini: II. Local and regional variation in patterns of resistance and racial structure. Evolution, 1991,45(7):1618-1627.
doi: 10.1111/j.1558-5646.1991.tb02667.x pmid: 28564135
[19] DUPLESSIS S, CUOMO C A, LIN Y C, AERTS A, TISSERANT E, VENEAULT-FOURREY C, JOLY D L, HACQUARD S, AMSELEM J, CANTAREL B L,et al. Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proceedings of the National Academy of Sciences of the United States of America, 2011,108(22):9166-9171.
doi: 10.1073/pnas.1019315108 pmid: 21536894
[20] STASKAWICZ B J, MUDGETT M B, DANGL J L, GALAN J E. Common and contrasting themes of plant and animal diseases. Science, 2001,292(5525):2285-2289.
[21] CUI H, TSUDA K, PARKER J E. Effector-triggered immunity: From pathogen perception to robust defense. Annual Review of Plant Biology, 2015,66:487-511.
pmid: 25494461
[22] BRUCE M, NEUGEBAUER K A, JOLY D L, MIGEON P, CUOMO C A, WANG S, AKHUNOV E, BAKKEREN G, KOLMER J A, FELLERS J P. Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat. Frontiers in Plant Science, 2014,4: Article 520.
pmid: 24454317
[23] HU Z, YAN C, LIU P, HUANG Z, MA R, ZHANG C, WANG R, ZHANG Y, MARTINON F, MIAO D, DENG H, WANG J, CHANG J, CHAI J. Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science, 2013,341(6142):172-175.
doi: 10.1126/science.1236381 pmid: 23765277
[24] CATANZARITI A M, DODDS P N, LAWRENCE G J, AYLIFFE M A, ELLIS J G. Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors. The Plant Cell, 2006,18(1):243-256.
pmid: 16326930
[25] 杨作民, 解超杰, 孙其信. 后条中32时期我国小麦条锈抗源之现状. 作物学报, 2003,29(2):161-168.
YANG Z M, XIE C J, SUN Q X. Situation of the sources of stripe rust resistance of wheat in the post-CY32 era in China. Acta Agronomica Sinica, 2003,29(2):161-168. (in Chinese)
[26] CUOMO C A, BAKKEREN G, KHALIL H B, PANWAR V, JOLY D, LINNING R, SAKTHIKUMAR S, SONG X, ADICONIS X, FAN L,et al. Comparative analysis highlights variable genome content of wheat rusts and divergence of the mating loci. G3: Genes, Genomes, Genetics, 2017,7(2):361-376.
doi: 10.1534/g3.116.032797 pmid: 27913634
[27] CATANZARITI A M, DODDS P N, VE T, KOBE B, ELLIS J G, STASKAWICZL B J. The AvrM effector from flax rust has a structured c-terminal domain and interacts directly with the M resistance protein. Molecular Plant-Microbe Interactions, 2010,23(1):49-57.
doi: 10.1094/MPMI-23-1-0049 pmid: 19958138
[28] DODDS P N, LAWRENCE G J, CATANZARITI A M, AYLIFFE M A, ELLIS J G. The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells. The Plant Cell, 2004,16(3):755-768.
doi: 10.1105/tpc.020040 pmid: 14973158
[29] GIRALDO M C, VALENT B. Filamentous plant pathogen effectors in action. Nature Reviews Microbiology, 2013,11:800-814.
doi: 10.1038/nrmicro3119
[30] VANDER MERVE M M, KINNEAR M W, BARRETT L G, DODDS P N, ERICSON L, THRALL P H, BURDON J J. Positive selection in AvrP4 avirulence gene homologues across the genus Melampsora. Proceedings of the Royal Society B: Biological Sciences, 2009,276(1669):2913-2922.
doi: 10.1098/rspb.2009.0328 pmid: 19457888
[31] ANDERSON C, KHAN M A, CATANZARITI A M, JACK C A, NEMRI A, LAWRENCE G J, UPADHYAYA N M, HARDHAM A R, ELLIS J G, DODDS P N, JONES D A. Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini. BMC Genomics, 2016,17(1):667.
doi: 10.1186/s12864-016-3011-9
[32] OH M, RHA G B, YOON J H, SUNWOO Y, HONG S H, PARK S D. RTP1, a rat homologue of adenovims E1A-associated Protein BS69, interacts with DNA topoisomerase II. Korean Journal of Biological Sciences, 2002,6(3):277-282.
doi: 10.1080/12265071.2002.9647663
[33] PETER B, JOLY D L, DUPLEESSIS S. Effector proteins of rust fungi. Frontiers in Plant Science, 2014, 5: Article 416.
doi: 10.3389/fpls.2014.00416 pmid: 25191335
[34] SALCEDO A, RUTTER W, WANG S, AKHUNOVA A, BOLUS S, CHAO S, ANDERSON N, DE SOTO M F, ROUSE M, SZABO L, BOWDEN R L, DUBCOVSKY J, AKHUNOVL E. Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99. Science, 2017,358(6370):1604-1606.
doi: 10.1126/science.aao7294 pmid: 29269474
[35]