Cloning and Functional Validation of the Spore Production Gene PsCON6 from Wheat Stripe Rust Fungus

doi:10.7668/hbnxb.20195885

Abstract

Abstract:

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici,is a major disease that severely threatens China's food security.Urediniospores are key agents for the reproduction,dissemination,and infection of the pathogen,but the regulatory mechanisms of sporulation-related genes remain unclear.This study aims to screen and validate the function of the candidate gene PsCON6,which is highly expressed during the early infection stage of P.striiformis,to provide new insights into its pathogenic mechanisms.The PsCON6 gene was obtained from P.striiformis via homologous cloning,and its expression pattern during early infection was analyzed using qRT-PCR.Bioinformatics technology analysis of the amino acid sequence,conserved domains and physicochemical properties of the PsCON6 protein.Barley Stripe mosaic virus host-induced gene silencing(BSMV-HIGS)was used to transiently silence PsCON6,followed by measurements of host reactive oxygen species accumulation,fungal hyphal length and area,and pathogen biomass.Subcellular localization of PsCON6 was determined through transient expression assays.PsCON6 was significantly upregulated during the early infection stage of P.striiformis.The encoded protein contained two conserved conidiation-specific protein 6 domains and consisted of 83 amino acids.After HIGS-mediated silencing of PsCON6,the level of reactive oxygen species in the host significantly increased,while the length and area of the fungal hyphae significantly decreased,whereas urediniospore production remained unaffected.Subcellular localization revealed that PsCON6 was localized to the cell membrane.PsCON6 likely participates in regulating hyphal growth in P.striiformis but does not directly influence urediniospore formation,suggesting potential functional redundancy.The research findings provide new targets for revealing the pathogenic mechanism of wheat stripe rust and lay the foundation for further in-depth analysis of its molecular mechanisms.

Key words: Wheat stripe rust, PsCON6, HIGS, Spore production gene

CLC Number:

LIU Beibei, SHEN Yuyang, DENG Feifei, CHEN Jianghua, LI Jin, LI Guangkuo, GAO Haifeng, LI Yue. Cloning and Functional Validation of the Spore Production Gene PsCON6 from Wheat Stripe Rust Fungus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 185-193. doi: 10.7668/hbnxb.20195885.

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Figures/Tables 9

Fig.1 Cloning of PsCON6 gene

Fig.2 Structural domain prediction and protein transmembrane structure prediction of PsCON6 protein A.Structural domain prediction of PsCON6;B.Transmembrane structure prediction of PsCON6.

Fig.3 Prediction of PsCON6 protein structure A.Predicted secondary structure of PsCON6;B.Predicted tertiary structure of PsCON6.

Fig.4 Multiple sequence alignment and phylogenetic tree analysis of Con6 protein in different species A.Multiple sequence alignment of PsCON6;B.Phylogenetic tree analysis of PsCON6.

Fig.5 Analysis of the expression characteristics of PsCON6 in the infection process of wheat inoculated with wheat stripe rust US.Uredospore.The experiment was repeated three times,the same lowercase letters at the same time point indicate no significant difference(P>0.05),while different lowercase letters at the same time point indicate significant difference(P<0.05).The same as Fig.6-8.

Fig.6 Detection of sporulation phenotype,silencing efficiency and biomass of leaves after transient silencing of PsCON6 gene by BSMV-HIGS technique A.Phenotype of leaves after PsCON6 gene silencing(Mock.Blank control;BSMV∶PDS.Positive control,BSMV∶γ.Negative control);B.Silencing efficiency of the PsCON6 gene;C.Biomass measurement of the PsCON6 gene.

Fig.7 Leaf DAB staining and active oxygen area detection after instantaneous silencing of PsCON6 gene by BSMV-HIGS technique A.DAB staining after silencing the PsCON6 gene(SV.Stomatal cavity;H₂O₂.Reactive oxygen species); B.Detection of reactive oxygen area after silencing the PsCON6 gene.

Fig.8 Leaf WGA staining and mycelial length and mycelial area detection after transient silencing of PsCON6 gene by BSMV-HIGS technique A.WGA staining after PsCON6 gene silencing(SV.Substomatal vesicle;IH.Infection hyphae;HMC.Haustorial mother cell); B.Measurement of hyphal length after PsCON6 gene silencing;C.Measurement of hyphal area after PsCON6 gene silencing.

Fig.9 Subcellular localization of PsCON6 encoding protein in tobacco leaves

References 27

[1]	甘露, 谢美娟, 卢振华, 李明, 丁博, 邱丽娜, 谢晓东, 王俊斌. 小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析[J]. 华北农学报, 2024, 39(5):1-8.doi:10.7668/hbnxb.20195063.
	Gan L, Xie M J, Lu Z H, Li M, Ding B, Qiu L N, Xie X D, Wang J B. Cloning and stress-resistance functional analysis of calcium-dependent protein kinase gene TaCDPK17 in wheat[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5):1-8.
[2]	李凯丽, 耿明状, 王胜, 郝维浩, 卢杰, 陈璨, 司红起. 安农1687抗条锈病候选基因TraesCS2A01G070700鉴定与分析[J]. 华北农学报, 2024, 39(1):150-155.doi:10.7668/hbnxb.20194425.
	Li K L, Geng M Z, Wang S, Hao W H, Lu J, Chen C, Si H Q. Identification and analysis on the resistance to stripe rust candidate gene of TraesCS2A01G070700 from wheat cultivar Annong 1687[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1):150-155.
[3]	Kokla A, Leso M, Šimura J, Wärdig C, Hayashi M, Nishii N, Tsuchiya Y, Ljung K, Melnyk C W. A long-distance inhibitory system regulates haustoria numbers in parasitic plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122(8):e2424557122.doi:10.1073/pnas.2424557122.
[4]	Xu Q, Tang C L, Wang L K, Zhao C C, Kang Z S, Wang X J. Haustoria arsenals during the interaction between wheat and Puccinia striiformis f.sp.tritici[J]. Molecular Plant Pathology, 2020, 21(1):83-94.doi:10.1111/mpp.12882.
[5]	Shen G J, Zhang J X, Lei Y T, Xu Y X, Wu J Q. Between-plant signaling[J]. Annual Review of Plant Biology, 2023,74:367-386.doi:10.1146/annurev-arplant-070122-015430.
[6]	王秋玲. 小麦条锈菌PsICL1及PsG6PDH1基因的克隆及功能研究[D]. 杨凌: 西北农林科技大学, 2014.
	Wang Q L. Cloning and functional study of PsICL1 and PsG6PDH1 genes from Puccinia striiformis[D]. Yangling: Northwest A&F University, 2014.
[7]	Adams T H, Wieser J K, Yu J H. Asexual sporulation in Aspergillus nidulans[J]. Microbiology and Molecular Biology Reviews, 1998, 62(1):35-54.doi:10.1128/mmbr.62.1.35-54.1998.
[8]	Busby T M, Miller K Y, Miller B L. Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes[J]. Genetics, 1996, 143(1):155-163.doi:10.1093/genetics/143.1.155.
[9]	Berlin V, Yanofsky C. Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa[J]. Molecular and Cellular Biology, 1985, 5(4):849-855.doi:10.1128/mcb.5.4.849-855.1985.
[10]	Krach E K, Skaro M, Wu Y, Arnold J. Characterizing the gene environment interaction underlying natural morphological variation in Neurospora crassa conidiophores using high-throughput phenomics and transcriptomics[J]. G3 Genes\|Genomes\|Genetics, 2022, 12(4):jkac050.doi:10.1093/g3journal/jkac050.
[11]	Madi L, Ebbole D J, White B T, Yanofsky C. Mutants of Neurospora crassa that alter gene expression and conidia development[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(13):6226-6230.doi:10.1073/pnas.91.13.6226.
[12]	Bailey-Shrode L, Ebbole D J. The fluffy gene of Neurospora crassa is necessary and sufficient to induce conidiophore development[J]. Genetics, 2004, 166(4):1741-1749.doi:10.1093/genetics/166.4.1741.
[13]	Kim S, Park S Y, Kim K S, Rho H S, Chi M H, Choi J, Park J, Kong S, Park J, Goh J, Lee Y H. Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae[J]. PLoS Genetics, 2009, 5(12):e1000757.doi:10.1371/journal.pgen.1000757.
[14]	Kuo C Y, Chen S A, Hsueh Y P. The high osmolarity glycerol(HOG)pathway functions in osmosensing,trap morphogenesis and conidiation of the nematode-trapping fungus Arthrobotrys oligospora[J]. Journal of Fungi, 2020, 6(4):191.doi:10.3390/jof6040191.
[15]	Wei C X, Wen C Y, Zhang Y Y, Du H Y, Zhong R R, Guan Z Z, Wang M J, Qin Y H, Wang F, Song L Y, Zhao Y. The FomYjeF protein influences the sporulation and virulence of Fusarium oxysporum f.sp.momordicae[J]. International Journal of Molecular Sciences, 2023, 24(8):7260.doi:10.3390/ijms24087260.
[16]	宋波. 禾谷镰刀菌突变体库的筛选以及Myo2基因的功能分析[D]. 武汉: 华中农业大学, 2013.doi:10.7666/d.D01407080.
	Song B. Screening of Fusarium graminearum mutant library and functional analysis of Myo2 gene[D]. Wuhan: Huazhong Agricultural University, 2013.
[17]	Guo L J, Wang J, Liang C C, Yang L Y, Zhou Y, Liu L, Huang J S. Fosp9,a novel secreted protein,is essential for the full virulence of Fusarium oxysporum f.sp.cubense on banana(Musa spp.)[J]. Applied and Environmental Microbiology, 2022, 88(6):e00604-21.doi:10.1128/aem.00604-21.
[18]	陈玥颖, 郭军, 代西维, 段迎辉, 魏国荣, 黄丽丽, 康振生. 小麦条锈菌一个产孢相关基因PsCon1的克隆及表达特征分析[J]. 中国农业科学, 2010, 43(6):1156-1163.doi:10.3864/j.issn.0578-1752.2010.06.007.
	Chen Y Y, Guo J, Dai X W, Duan Y H, Wei G R, Huang L L, Kang Z S. Cloning and expression analysis of a conidiation-related gene PsCon1 from Puccinia striiformis f.sp.tritici[J]. Scientia Agricultura Sinica, 2010, 43(6):1156-1163.
[19]	Kim T, Lee S H, Oh Y T, Jeon J. A histone deacetylase,MoHDA1 regulates asexual development and virulence in the rice blast fungus[J]. The Plant Pathology Journal, 2020, 36(4):314-322.doi:10.5423/ppj.oa.06.2020.0099.
[20]	Ruiz-Roldán M C, Maier F J, Schäfer W. PTK1,a mitogen-activated-protein kinase gene,is required for conidiation,appressorium formation,and pathogenicity of Pyrenophora teres on barley[J]. Molecular Plant-Microbe Interactions, 2001, 14(2):116-125.doi:10.1094/mpmi.2001.14.2.116.
[21]	Lu Q, Lu J P, Li X D, Liu X H, Min H, Lin F C. Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination[J]. Journal of Zhejiang University Science B, 2008, 9(7):511-519.doi:10.1631/jzus.B0820015.
[22]	Moriwaki A, Kihara J, Mori C, Arase S. A MAP kinase gene,BMK1,is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae[J]. Microbiological Research, 2007, 162(2):108-114.doi:10.1016/j.micres.2006.01.014.
[23]	代西维. 小麦条锈菌PsMAPK1,PsCdc2,PsFuz7基因的克隆及PsMAPK1基因功能验证[D]. 杨凌: 西北农林科技大学, 2010.
	Dai X W. Cloning of PsMAPK1,PsCdc2 and PsFuz7 genes from Puccinia striiformis and functional verification of PsMAPK1 gene[D]. Yangling: Northwest A&F University, 2010.
[24]	Qin J, Zhao P C, Ye Z Q, Sun L F, Hu X P, Zhang J. Chitin synthase genes are differentially required for growth,stress response,and virulence in Verticillium dahliae[J]. Journal of Fungi, 2022, 8(7):681.doi:10.3390/jof8070681.
[25]	蒋金河. 镰刀菌几丁质合成酶Chs1和Chs2基因小片段RNA干扰功能分析[D]. 武汉: 华中农业大学, 2015.doi:10.7666/d.Y2803218.
	Jiang J H. Analysis of RNA interference function of small fragments of chitin synthase Chs1 and Chs2 genes in Fusarium[D]. Wuhan: Huazhong Agricultural University, 2015.
[26]	Zhao M X, Zhang Y H, Guo H L, Gan P F, Cai M M, Kang Z S, Cheng Y L. Identification and functional analysis of cap genes from the wheat stripe rust fungus Puccinia striiformis f.sp.tritici[J]. Journal of Fungi, 2023, 9(7):734.doi:10.3390/jof9070734.
[27]	刘枭. 大豆疫霉超氧化物歧化酶基因(PsSOD1)的功能分析[D]. 合肥: 安徽农业大学, 2021.doi:10.26919/d.cnki.gannu.2021.000192.
	Liu X. Functional analysis of superoxide dismutase gene(PsSOD1) of Phytophthora sojae[D]. Hefei: Anhui Agricultural University, 2021.

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