A virulent milRNA of Fusarium oxysporum f. sp. cubense impairs plant resistance by targeting banana AP2 transcription factor coding gene MaPTI6L

Abstract

Fungi produce microRNA-like RNAs (milRNAs) with functional importance in various biological processes. Our previous research identified a new milRNA Foc-milR87 from Fusarium oxysporum f. sp. cubense, which contributes to fungal virulence by targeting the pathogen glycosyl hydrolase encoding gene. However, the potential roles of fungal milRNAs in interactions with hosts are not well understood. This study demonstrated that Foc-milR87 specifically suppressed the expression of MaPTI6L, a pathogenesis-related gene that encodes a transcriptional activator in the banana (Musa acuminata Cavendish group cv. ‘Baxi Jiao’) genome, by targeting the 3'untranslated region (UTR) of MaPTI6L. Transient overexpression of MaPTI6L activated plant defense responses that depend on its nuclear localization, yet co-expression with Foc-milR87 attenuated these responses. MaPTI6L enhanced plant resistance by promoting transcription of the salicylic acid signaling pathway marker gene MaEDS1. Sequence analysis of the MaPTI6L gene in 19 banana varieties, particularly those resistant to Fusarium wilt, uncovered single nucleotide polymorphisms (SNPs) at Foc-milR87 target sites. Experimental validation showed that these SNPs significantly reduce the microRNA's ability to suppress target gene expression. Our findings reveal that Foc-milR87 plays an important role in impairing plant resistance by targeting MaPTI6L mRNA and reducing MaEDS1 transcription during the early infection stage, suggesting the 3'UTR of MaPTI6L as a promising target for genome editing in generation of disease-resistant banana cultivars.

Acknowledgements

We thank Dr. Bingzhi Huang (Institution of Fruit Tree Research, Guangdong Academy of Agricultural Sciences) for the different banana varieties’ support. We also thank Professor Shaobin Zhong (North Dakota State University), Hai Zhou, and Yi-zhen Deng (South China Agricultural University) for helpful suggestions regarding this manuscript. This work was financially supported by the National Natural Science Foundation of China (31871911), the Natural Science Foundation of Guangdong Province (2023A1515012965), and the earmarked fund for China Agricultural Research System (CARS-31).

Author Contributions

M.L., Z.J., and H.L. designed the study. J.Z., J.S., C.H., and J.H. performed the experiments. J.Z. and M.L. analyzed the data. Z.J., J.S., G.K., and M.L. wrote the manuscript.

Data Availability

All data in this study were provided in the article and its supplementary materials.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Ordonez N, Seidl M, Waalwijk C. et al. Worse comes to worst: bananas and Panama disease-when plant and pathogen clones meet. PLoS Pathog. 2015; 11:e1005197

[2] Ploetz R. Fusarium wilt of banana. Phytopathology. 2015; 105: 1512-21

[3] Zheng S, García-Bastidas F, Li X. et al. New geographical insights of the latest expansion of fusarium oxysporum f.sp. cubense trop-ical race 4 into the greater mekong subregion. Front Plant Sci. 2019; 9:457

[4] Viljoen A, Ma J, Molina A. Fusarium wilt (Panama disease) and monoculture banana production:resurgence of a century-old disease. In: RistainoJB, RecordsA.eds. Emerging Plant Diseases and Global Food Security. APS Press: St Paul, 2020,159-84

[5] Zorrilla-Fontanesi Y, Pauwels L, Panis B. et al. Strategies to revise agrosystems and breeding to control Fusarium wilt of banana. Nat Food. 2020; 1:599-604

[6] Li H, Li Y, Nie Y. Research status of occurrence and control of fusarium wilt of banana. J South China Agric Univ. 2019; 40:128-36

[7] Tripathi L, Ntui V, Tripathi J. CRISPR/Cas9-based genome editing of banana for disease resistance. Curr Opin Plant Biol. 2020; 56: 118-26

[8] Dale J, James A, Paul J. et al. Transgenic cavendish bananas with resistance to fusarium wilt tropical race 4. Nat Commun. 2017; 8:1496

[9] Dou T, Shao X, Hu C. et al. Host-induced gene silencing of Foc TR4 ERG6/11 genes exhibits superior resistance to Fusarium wilt of banana. Plant Biotechnol J. 2020; 18:11-3

[10] Maxmen A. CRISPR could save bananas from fungus. Nature. 2019; 574:15

[11] Lee H, Li L, Gu W. et al. Diverse pathways generate microRNA-like RNAs and dicer-independent small interfering RNAs in fungi. Mol Cell. 2010; 38:803-14

[12] Weiberg A, Wang M, Lin F. et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science. 2013; 342:118-23

[13] Jin Y, Zhao J, Zhao P. et al. A fungal milRNA mediates epigenetic repression of a virulence gene in Verticillium dahliae. Philos Trans RSoc B. 2019; 374:20180309

[14] Wang M, Weiberg A, Dellota E. et al. Botrytis small RNA Bc-siR37 suppresses plant defense genes by cross-kingdom RNAi. RNA Biol. 2017; 14:421-8

[15] Xu M, Li G, Guo Y. et al. A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silenc-ing two host receptor-like kinase genes. New Phytol. 2022; 233: 2503-19

[16] Ji H, Mao H, Li S. et al. Fol-milR1, a pathogenicity factor of fusarium oxysporum, confers tomato wilt disease resistance by impairing host immune responses. New Phytol. 2021; 232:705-18

[17] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta. 2012; 1819:86-96

[18] Licausi F, Ohme-Takagi M, Perata P. APETALA2/ethylene respon-sive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol. 2013; 199: 639-49

[19] Müller M, Munné-Bosch S. Ethylene response factors: a key regulatory hub in hormone and stress signaling. Plant Physiol. 2015; 169:32-41

[20] Feng K, Hou X, Xing G. et al. Advances in AP2/ERF super-family transcription factors in plant. Crit Rev Biotechnol. 2020; 40:750-76

[21] Hu D, Yao Y, Lv Y. et al. The OsSRO1c-OsDREB2B complex undergoes protein phase transition to enhance cold tolerance in rice. Mol Plant. 2024; 17:1520-38

[22] Lim C, Kang K, Lim J. et al. RICE LONG GRAIN 3 delays dark-induced senescence by downregulating abscisic acid signaling and upregulating reactive oxygen species scavenging activity. Plant J. 2024; 120:1474-8

[23] Liu A, Cheng C. Pathogen-induced ERF68 regulates hypersensi-tive cell death in tomato. Mol Plant Pathol. 2017; 18:1062-74

[24] Zhao Y, Chang X, Qi D. et al. A novel soybean ERF transcrip-tion factor, GmERF113, increases resistance to Phytophthora sojae infection in soybean. Front Plant Sci. 2017; 8:299

[25] Li M, Xie L, Wang M. et al. FoQDE2-dependent milRNA pro-motes Fusarium oxysporum f. sp. cubense virulence by silencing a glycosyl hydrolase coding gene expression. PLoS Pathog. 2022; 8: e1010157

[26] Zhou J, Tang X, Martin G. The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes. EMBO J. 1997; 16: 3207-18

[27] Mittler R, Zandalinas S, Fichman Y. et al. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol. 2022; 23: 663-79

[28] Chowdhury J, Schober M, Shirley N. et al. Down-regulation of the glucan synthase-like 6 gene (HvGsl6) in barley leads to decreased callose accumulation and increased cell wall penetration by Blumeria graminis f. sp. hordei. New Phytol. 2016; 212:434-43

[29] Voigt C. Cellulose/callose glucan networks: the key to powdery mildew resistance in plants? New Phytol. 2016; 212:303-5

[30] Wang H, Song S, Gao S. et al. The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis. Plant Cell. 2024; 36: 427-46

[31] Huang CY, Wang H, Hu P. et al. Small RNAs-big players in plant-microbe interactions. Cell Host and Microbe. 2019; 26:173-82

[32] Zhao JH, Zhang T, Liu QY. et al. Trans-kingdom RNAs and their fates in recipient cells: advances, utilization, and perspectives. Plant Commun. 2021; 2:100167

[33] Rong FR, Lv YS, Deng PC. et al. Switching action modes of miR408-5 p mediates auxin signaling in rice. Nat Commun. 2024; 15:2525

[34] Jeseniˇcnik T, ˇStajner N, Radiˇsek S. et al. Discovery of microRNA-like small RNAs in pathogenic plant fungus Verticillium nonalfal-fae using high-throughput sequencing and qPCR and RLM-RACE validation. Int J Mol Sci. 2022; 23:900

[35] Bogdanove A, Martin G. AvrPto-dependent Pto-interacting pro-teins and AvrPto-interacting proteins in tomato. Proc Natl Acad Sci USA. 2000; 97:8836-40

[36] Zhou J, Loh Y, Bressan R, et al. The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response. Cell. 1995; 83:925-35

[37] Gu Y, Wildermuth M, Chakravarthy S. et al. Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis. Plant Cell. 2002; 14:817-31

[38] Wang Y, Feng G, Zhang Z. et al. Overexpression of Pti4, Pti5, and Pti 6 in tomato promote plant defense and fruit ripening. Plant Sci. 2021; 302:110702

[39] He P, Warren R, Zhao T. et al. Overexpression of Pti5 in tomato potentiates pathogen-induced defense gene expression and enhances disease resistance to Pseudomonas syringae pv. Tomato. Mol Plant-Microbe Interact. 2001; 14:1453-7

[40] Gu Y, Yang C, Thara V. et al. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase. Plant Cell. 2000; 12:771-86

[41] Sun M, Qiu L, Liu Y. et al. Pto interaction proteins: critical regulators in plant development and stress response. Front Plant Sci. 2022; 13:774229

[42] Su K, Guo Y, Zhao Y. et al. Candidate genes for grape white rot resistance based on SMRT and Illumina sequencing. BMC Plant Biol. 2019; 19:501

[43] Chen H, Li M, Qi G. et al. Two interacting transcriptional coacti-vators cooperatively control plant immune responses. Sci Adv. 2021; 7:eabl7173

[44] Robert-Seilaniantz A, Grant M, Jones J. Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev Phytopathol. 2011; 49:317-43

[45] Song N, Ma L, Wang W. et al. An ERF2-like transcription factor regulates production of the defense sesquiterpene capsidiol upon Alternaria alternata infection. JExp Bot. 2019; 70:5895-908

[46] Padma T, Pradeep K, Arpit C. et al. Molecular insights into the variability and pathogenicity of Fusarium odoratissimum,the causal agent of Panama wilt disease in banana. Microb Pathog. 2024; 190:106594

[47] Li W, Li P, Deng Y. et al. A plant cell death-inducing protein from litchi interacts with Peronophythora litchii pectate lyase and enhances plant resistance. Nat Commun. 2024; 15:22

[48] Wang Z, Yang B, Zheng W. et al. Recognition of glycoside hydrolase 12 proteins by the immune receptor RXEG1 confers Fusarium head blight resistance in wheat. Plant Biotechnol J. 2023; 21:769-81

[49] Wang Y, Pruitt R, Nürnberger T. et al. Evasion of plant immunity by microbial pathogens. Nat Rev Microbiol. 2022; 20:449-64

[50] Li M, Yang B, Leng Y. et al. Molecular characterization of Fusarium oxysporum f. sp. cubense race 1 and 4 isolates from Taiwan and Southern China. Can J Plant Pathol. 2011; 33:168-78

[51] Martínez-Soto D, Yu H, Allen KS. et al. Differential colonization of the plant vasculature between endophytic versus pathogenic Fusarium oxysporum strains. Mol Plant-Microbe Interact. 2023; 36: 4-13

[52] Li M, Xie X, Lin X. et al. Functional characterization of the gene FoOCH1 encoding a putative α-1, 6-mannosyltransferase in Fusarium oxysporum f. sp. cubense. Fungal Genet Biol. 2014; 65:1-13

[53] Chen H, Feng Q, Xu C. et al. Screening of banana clones for resistance to Fusarium wilt (Fusarium oxysporum f. sp. cubense). J South China Agric Univ. 2006; 27:9-12

[54] Xu L, Zhang X, Gan D. et al. Introduction and trial planting of ‘Haigong Jiao’ (Musa AA). Chin J Trop Agric. 2013; 33:24-8

[55] Choi H, Kim Y, Hwang B. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associ-ated with disease and plant immunity. Mol Plant-Microbe Interact. 2011; 24:68-78

[56] Zhang L, Ni H, Du X. et al. The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fun-gal infections. New Phytol. 2017; 215:368-81

[57] Peng H, Pu Y, Yang X. et al. Overexpression of a pathogenesis-related gene NbHIN1 confers resistance to tobacco mosaic virus in Nicotiana benthamiana by potentially activating the jasmonic acid signaling pathway. Plant Sci. 2019; 283:147-56

[58] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870-4

[59] He J, Zhong J, Jin L. et al. A virulent milRNA inhibits host immunity by silencing a host receptor-like kinase MaLYK3 and facilitates infection by fusarium oxysporum f. sp. cubense. Mol Plant Pathol. 2024; 25:e70016

[60] Situ J, Jiang L, Fan X. et al. An RXLR effector PlAvh142 from Peronophythora litchii triggers plant cell death and contributes to virulence. Mol Plant Pathol. 2020; 21:415-28

[61] Huang H, Liu X, Arshad R. et al. Telomere-to-telomere haplotype-resolved reference genome reveals subgenome divergence and disease resistance in triploid cavendish banana. Hortic Res. 2023; 10:uhad153

[62] Lescot M, Déhais P, Thijs G. et al.PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30:325-7

[63] Liu Y, An J, Gao N. et al. MdTCP46 interacts with MdABI5 to neg-atively regulate ABA signaling and drought response in apple. Plant Cell Environ. 2022; 45:3233-48