Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

doi:10.16420/j.issn.0513-353x.2021-1081

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (2): 279-294.doi: 10.16420/j.issn.0513-353x.2021-1081

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Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

ZHANG Xin, QI Yanxiang, ZENG Fanyun, WANG Yanwei, XIE Peilan, XIE Yixian^*(), PENG Jun^*()

Key Laboratory of Integrated Pest Management on Tropical Crops，Ministry of Agriculture and Rural Affairs，Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests，Environment and Plant Protection Institute，Chinese Academy of Tropical Agricultural Sciences，Haikou 571101，China

Received:2022-06-16 Revised:2022-09-27 Online:2023-02-25 Published:2023-03-06
Contact: *（E-mail：yixian81@126.com，swaupj2@126.com）

Abstract

Abstract:

The fungal pathogen Fusarium oxysporum f. sp. cubense（Foc）is an important soil-borne pathogenic fungus which causes Banana Fusarium wilt. The Foc race 4（Foc 4）attacking almost all Cavendish cultivars is currently noteworthy in global banana production. In order to explore the function of DCL-like genes in Foc4，the gene-knockout mutants ΔFocDCL1，ΔFocDCL2 and ΔFocDCL1/2 were obtained by homologous recombination technique. The phenotype analysis showed the mutants showed no significant differences on vegetative growth but significant reduced in conidia production compared to the Foc4. The ΔFocDCL2 mutant increased sensitive to Congo red，and the mutant significantly reduced the size of the colony and increased the number of aerial hyphae after Congo red treatment. Next，the pathogenicity test showed that virulence of ΔFocDCL2 and ΔFocDCL1/2 were significantly reduced compared with Foc4，respectively. Furthermore，miRNA deep sequencing data showed the length distribution of the total reads，and the frequency of the 5′ first nucleotide bias of the mutants were markedly changed compared with Foc4，respectively. The miRNA could be generated depending on individual，alternative or joint DCL manners，indicating the FocDCL with overlapped and redundant function in small RNA biogenesis. In addition，DCLs-independent miRNA also be identified in the sequencing data. These results indicated that FocDCL function involved in the stress response，conidia，pathogenicity and generation of miRNAs in Foc4.

Key words: banana, Fusarium wilt, Fusarium oxysporum f. sp. cubense, Dicer-like gene, pathogenicity test, miRNA

CLC Number:

S668.1

ZHANG Xin, QI Yanxiang, ZENG Fanyun, WANG Yanwei, XIE Peilan, XIE Yixian, PENG Jun. Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4[J]. Acta Horticulturae Sinica, 2023, 50(2): 279-294.

Figures/Tables 13

Table 1 The FocDCLs single and double gene knock-out primers used in this study

引物 Primer	序列（5′-3′） Sequence	引物 Primer	序列（5′-3′） Sequence
HYG-F	CTTGGCTGGAGCTAGTGGAGGT	NEO-F	TCTAGATTAACGCTTACAATTTCC
HYG-R DCL1-LBCK	CCCGGTCGGCATCTACTCTATTC CCAGGCTATGGTCCCAAGAA	NEO-R DCL2-NEO-LBCK	TCAGAAGAACTCGTCAAGAAGG CAGCAGATGTAATAGTCGCCG
DCL1-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGCAAGAGTCCGCTACAATCTCAA	DCL2-NEO--LB-R	GAAATTGTAAGCGTTAATCTAGCCTTGATGCCCTCCTTATCC
DCL1-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGAGCGTTAGAAGCGTAGACAA	DCL2-NEO-RB-F	CCTTCTTGACGAGTTCTTCTGATTGAGAGTGCGGAGGGACTG
DCL1-RBCK	CAACAAACAAGACCTCCTCTC	DCL2-NEO-RBCK	GAGGGTGAGATGAACGGTGA
DCL1-LB-F	GCAAAGAGTCTATCGTGTGAGCC	DCL2-NEO-LB-F	CGACTTACACAAATACATCCTCCC
HYG-R1	GGATGCCTCCGCTCGAAGTA	NEO-R1	GAGCAAGGTGAGATGACAGGAG
HYG-F1	CGTTGCAAGACCTGCCTGAA	NEO-F1	CACCACTCGATCCGTCACCAAC
DCL1-RB-R	CGGTAAAGGATTGGGATTGTTG	DCL2-NEO-RB-R	CGTCTTTGTCTCCATCAACTTCG
HPT-LBCK	GACAGACGTCGCGGTGAGTT	NEO-LBCK	GAATGTCGTCAAGCGGGAAC
HPT-RBCK	TCTGGACCGATGGCTGTGTAG	NEO-RBCK	CGACCACCAAGCGAAACATC
DCL1-1784F	GTCTCTCTCTTTCTGCTGACCG	pFCC1-F1-XhoI	AATTCTCGAGAATTGATACGGCTGGCGAAG
DCL1-3031R	TCAAGGCTGGGATTCAACTTAC	pFCC1-R1-HindⅢ	AGAAAGCTTTCTCAACACCAAGGCCAGT
DCL2-LBCK	AGCATTCGTCAACTTTGCCA	pFCC1-F2-BglII	GGCAGATCTTCTCAACACCAAGGCCAGT
DCL2-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGTCCATCAGCACTCACATCACTC	pFCC1-R2-KpnI FCC1-qRT-F	CCAGGTACCAATTGATACGGCTGGCGAAG TCGACAGCAACGTGGAGATT
DCL2-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGGCATCACTAAACACTCCTCCTTGT	FCC1-qRT-R DCL1-qRT-F	ACCTGTTGATCTGTTCGCGA AGAACAAGTCCTGGCTCTCC
DCL2-RBCK	GAGGGTGAGATGAACGGTGAC	DCL1-qRT-R	GTCGCAATCTGGAACGTCGA
DCL2-LB-F	ACGCTTGGAGAGAATGCGAG	DCL2-qRT-F	TCGATGGAGTTGTGGAGTCA
DCL2-RB-R	AGCCATCAGTCGTAAGAGCAA	DCL2-qRT-R	GCATTCTCCGCAGCTTTGGT
DCL2-988F	CACCTCATTCGCTCACTCTACG	qFocUBI-1F	CCAACCCTGACGATCCTCTTGTGC
DCL2-2373R	ATCACAACCCGACTTCCAGC	qFocUBI-1R	TACTTTCGAGTCCACTCCCGAGCTG

Fig. 1 FocDCL1（left）and FocDLC2（right）gene knockout and PCR screening the positive mutants A，F：The schematic map of single-gene knockout，the arrows indicate the position of the primers，HY and YG indicate the first and second part of HYG resistance gene，respectively；B，G：Amplification of up and down recombinant from mutants by PCR；C，H：Amplification of endogenous gene by PCR；D，I：Amplification of Hygromycin B resistance gene by PCR；E，J：qRT-PCR detection of DCL1 and DCL2.

Fig. 2 The schematic diagram of ΔFocDCL1/2 double knockout and the detection of its mutant gene A：The schematic diagram of ΔFocDCL1/2 double knockout. The arrows indicate the position of the primers，Ne and EO indicate the first and second part of NEO resistance gene，respectively；B：Amplification of HYG and NEO by PCR；C：Amplification of FocDCL1 and FocDCL2 by PCR；D：Amplification of up and down recombinant from mutants by PCR；E：qRT-PCR detection of DCL1 and DCL2.

Fig. 3 The phylogenetic tree and conserved domain of FocDCL Neighbor-joining method was used with 1 000 bootstrap replicates.

Fig. 4 The phenotype of FocDCL gene knockout mutants Different lowercase letters indicate significant differences（P < 0.05）. The same below.

Fig. 5 Colony morphology of FocDCL knockout mutants on MM medium containing abiotic stress reagents

Fig. 6 Growth inhibition rate of FocDCL knockout mutants on MM medium containing abiotic stress reagents

Fig. 7 Symptoms of banana leaves and corms after inoculation with FocDCLs knockout mutants Disease index in parentheses. Different lowercase letters indicate significant difference（P < 0.05）.

Fig. 8 Phenotypic analysis of hpRNAi-FCC1 induced gene silencing in ΔFocDCL1 and ΔFocDCL2 ΔFocFCC1 with red colony phenotypes served as positive control.

Fig. 9 The relative mRNA expression level of FCC1 in silencing transformant

Table 2 The statistics of small RNA sequence date and miRNAs of DCL knockout mutants

样品 Sample ID	总数量 Total reads	匹配数量 Mapped reads	已知miRNA Known-miRNA	新的miRNA Novel-miRNA	靶标基因 Target gene
Foc4	18 931 444	6 494 076	16	39	3 829
ΔFocDCL1	8 922 109	2 851 863	10	33	1 772
ΔFocDCL2	9 046 433	3 373 316	9	14	1 105
ΔFocDCL1/2	19 831 731	11 454 808	6	11	818

Fig. 10 Statistics analysis of small RNA sequence from FocDCL knockout mutants A：The length distribution of unique reads of sRNA. B：Nucleotide frequency of the 5′ end of small RNAs；C：Statistics of specific miRNA.

Fig. 11 The miRNA comparison between FocDCL knockout mutants and Foc4（A）and identification of FocDCL-dependent miRNAs（B）

References 24

[1]	Allen E, Xie Z, Gustafson A M, Carrington J C. 2005. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121 (2):207-221. doi: 10.1016/j.cell.2005.04.004 pmid: 15851028
[2]	Carreras-Villaseñor N, Esquivel-Naranjo E U, Villalobos-Escobedo J M, Abreu-Goodger C, Herrera-Estrella A. 2013. The RNAi machinery regulates growth and development in the filamentous fungus Trichoderma atroviride. Molecular Microbiology, 89 (1):96-112. doi: 10.1111/mmi.12261 pmid: 23672609
[3]	Chen Y, Gao Q, Huang M, Liu Y, Liu Z, Liu X, Ma Z. 2015. Characterization of RNA silencing components in the plant pathogenic fungus Fusarium graminearum. Scientific Reports, 5:12500. doi: 10.1038/srep12500 pmid: 26212591
[4]	Choi J, Kim K T, Jeon J, Wu J, Song H, Asiegbu F O, Lee Y H. 2014. funRNA:a fungi-centered genomics platform for genes encoding key components of RNAi. BMC Genomics, 15 (Suppl 9):S14.
[5]	Feng H, Xu M, Liu Y, Dong R, Gao X, Huang L. 2017. Dicer-like genes are required for H₂O₂ and KCl stress responses,pathogenicity and small RNA generation in Valsa mali. Frontiers in Microbiology, 8:1166. doi: 10.3389/fmicb.2017.01166 pmid: 28690605
[6]	Gaffar F Y, Imani J, Karlovsky P, Koch A, Kogel K H. 2019. Different components of the RNA interference machinery are required for conidiation,ascosporogenesis,virulence,deoxynivalenol production,and fungal inhibition by exogenous double-stranded RNA in the head blight pathogen Fusarium graminearum. Frontiers in Microbiology, 10:1662. doi: 10.3389/fmicb.2019.01662 URL
[7]	Hwang I S, Ahn I P. 2016. Multi-homologous recombination-based gene manipulation in the rice pathogen Fusarium fujikuroi. Plant Pathology Journal, 32 (3):173-181. doi: 10.5423/PPJ.OA.12.2015.0263 pmid: 27298592
[8]	Jin Y, Zhao J H, Zhao P, Zhang T, Wang S, Guo H S. 2019. A fungal milRNA mediates epigenetic repression of a virulence gene in Verticillium dahliae. Philosophical Transactions B, 374 (1767):20180309. doi: 10.1098/rstb.2018.0309 URL
[9]	Kadotani N, Nakayashiki H, Tosa Y, Mayama S. 2004. One of the two Dicer-like proteins in the filamentous fungi Magnaporthe oryzae genome is responsible for hairpin RNA-triggered RNA silencing and related small interfering RNA accumulation. Journal of Biological Chemistry, 279 (43):44467-44474. doi: 10.1074/jbc.M408259200 pmid: 15304480
[10]	Lee H C, Li L, Gu W, Xue Z, Crosthwaite S K, Pertsemlidis A, Lewis Z A, Freitag M, Selker E U, Mello C C, Liu Y. 2010. Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Molecualr Cell, 38 (6):803-814.
[11]	Liang L Q. 2014. Function analysisi of Dicer,Argonaute and Rdrp involved in small RNAs biogenesis in Fusarium oxysporum[Ph. D. Dissertation]. Beijing: China Agricultural University. (in Chinese)
	梁丽琴. 2014. 尖孢镰刀菌小RNA发生相关Dicer,Argonaute和Rdrp基因功能研究[博士论文]. 北京: 中国农业大学.
[12]	Luo He, Li Weijia, Li He, Zhang Zhihong. 2020. FaRGA1gene silencing changes the characteristics of flowering and runner producing in strawberry. Acta Horticulturae Sinica, 47 (12):2331-2339. (in Chinese)
	罗贺, 李伟佳, 李贺, 张志宏. 2020. 草莓FaRGA1基因沉默改变开花和匍匐茎抽生特性. 园艺学报, 47 (12):2331-2339.
[13]	Macrae I J, Zhou K, Li F, Repic A, Brooks A N, Cande W Z, Adams P D, Doudna J A. 2006. Structural basis for double-stranded RNA processing by Dicer. Science, 311 (5758):195-198. doi: 10.1126/science.1121638 pmid: 16410517
[14]	Mohamed A A, Mak C, Liew K W, Ho Y W. 1999. Early evaluation of banana plants at nursery stage for fusarium wilt tolerance //Molina A B,Nik Masdek N H,Liew K W. Seminar on banana Fusarium wilt management towards sustainable cultivation,Pahang,Malaysia. Montpellier:International Network for the Improvement of Banana and Plantain:174-185.
[15]	Nakayashiki H, Hanada S, Nguyen B Q, Naoki K, Yukio T, Shigeyuki M. 2005. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genetics and Biology, 42 (4):275-283. doi: 10.1016/j.fgb.2005.01.002 pmid: 15749047
[16]	Nicolás F E, de Haro J P, Torres-Martínez S, Ruiz-Vázquez R M. 2007. Mutants defective in a Mucor circinelloides Dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genetics and Biology, 44 (6):504-516. doi: 10.1016/j.fgb.2006.09.003 URL
[17]	Schirawski J, Mannhaupt G, Münch K, Brefort T, Schipper K, Doehlemann G, Di Stasio M, Rössel N, Mendoza-Mendoza A, Pester D, Müller O, Winterberg B, Meyer E, Ghareeb H, Wollenberg T, Münsterkötter M, Wong P, Walter M, Stukenbrock E, Güldener U, Kahmann R. 2010. Pathogenicity determinants in smut fungi revealed by genome comparison. Science, 330 (6010):1546-1548. doi: 10.1126/science.1195330 pmid: 21148393
[18]	Schultz J, Milpetz F, Bork P, Ponting C P. 1998. SMART,a simple modular architecture research tool: identification of signaling domains. Proceedings of the National Academy of Sciences of the United States of America, 95 (11):5857-5864.
[19]	Shim W B, Woloshuk C P. 2001. Regulation of fumonisin B(1) biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like(C-type)gene,FCC1. Applied and Environmental Microbiology, 67 (4):1607-1612. pmid: 11282612
[20]	Siamak S B, Zheng S J. 2018. Banana fusarium wilt(Fusarium oxysporum f. sp. cubense)control and resistance,in the context of developing wilt-resistant bananas within sustainable production systems. Horticultural Plant Journal, 4 (5):208-218. doi: 10.1016/j.hpj.2018.08.001 URL
[21]	Wang M, Jin H. 2017. Spray-induced gene silencing:a powerful innovative strategy for crop protection. Trends in Microbiology, 25 (1):4-6. doi: 10.1016/j.tim.2016.11.011 URL
[22]	Wang W, Tang W. 2018. Generation of Fusarium graminearum knockout mutants by the split-marker recombination approach. Bio-protocol, e2976. doi:10.21769/BioProtoc.2976. doi: 10.21769/BioProtoc.2976
[23]	Xue Z, Yuan H, Guo J, Liu Y. 2012. Reconstitution of an Argonaute-dependent small RNA biogenesis pathway reveals a handover mechanism involving the RNA exosome and the exonuclease QIP. Molecular Cell, 46 (3):299-310. doi: 10.1016/j.molcel.2012.03.019 pmid: 22516970
[24]	Yang Xingyu, Xu Linbing, Wu Yuanli, Qiu Diyang, Fan Linlin, Huang Bingzhi. 2022. Genome(ABBB)identification of a hybrid banana cultivar‘Fenza 1’. Acta Horticulturae Sinica, 49 (9):1991-1997. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0514
	杨兴玉, 许林兵, 吴元立, 邱迪洋, 范琳琳, 黄秉智. 2022. 香蕉杂交种‘粉杂1号’ABBB基因组鉴定. 园艺学报, 49 (9):1991-1997. doi: 10.16420/j.issn.0513-353x.2021-0514

Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

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