The endocytic pathway for absorption of exogenous RNAs in Verticillium dahliae

Abstract

RNAi technologies have been exploited to control viruses, pests, oomycetes, and fungal phytopathogens that cause disasters in host plants, including many agronomically significant crops. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) has been applied as a trigger for trans-kingdom RNAi between hosts and fungi. However, it is unclear what process mediates RNA uptake by fungi. In this study, by using live-cell imaging, we determined that exogenously synthesized RNA or small RNA (sRNA) was indiscriminately absorbed into Verticillium dahliae, a notorious pathogenic fungus. Moreover, the application of endocytic inhibitors or deletion of endocytic-related genes reduced RNA uptake efficiency, showing that RNA absorption by fungal cells occurs mainly through endocytosis. In addition, we found that the endocytosed fluorescence-labeled RNAs were partly colocalized with endosome marker genes. Overall, our research concluded that exogenous RNA could be assimilated by V. dahliae through the endocytic pathway. Unraveling this cytological mechanism underlying trans-kingdom RNAi holds significant importance, especially considering the fact that RNAi-based strategies targeting pathogenic fungi are increasingly prevalent in the realm of crop protection.

Keywords

endocytic pathway / pathogenic fungi / RNA uptake / small RNA

References

[1] Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science. 2013; 342:118–123.

[2] Wang M, Weiberg A, Lin FM, Thomma BPHJ, Huang HD, Jin H. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nat Plants. 2016; 2:16151.

[3] Zhang BS, Li YC, Guo HS, Zhao JH. Verticillium dahliae secretes small RNA to target host MIR157d and retard plant floral transition during infection. Front Plant Sci. 2022; 13:847086.

[4] Wang B, Sun Y, Song N, Zhao M, Liu R, Feng H, et al. Puccinia striiformis f. sp. tritici microRNA-like RNA 1 (Pst-milR1), an important pathogenicity factor of Pst, impairs wheat resistance to Pst by suppressing the wheat pathogenesis-related 2 gene. New Phytol. 2017; 215:338–350.

[5] Zhang T, Zhao YL, Zhao JH, Wang S, Jin Y, Chen ZQ, et al. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat Plants. 2016; 2:16153.

[6] Ren B, Wang X, Duan J, Ma J. Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation. Science. 2019; 365:919–922.

[7] Shahid S, Kim G, Johnson NR, Wafula E, Wang F, Coruh C, et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 2018; 553:82–85.

[8] Nunes CC, Dean RA. Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies. Mol Plant Pathol. 2012; 13:519–529.

[9] Wang M, Jin H. Spray-induced gene silencing: a powerful innovative strategy for crop protection. TIM. 2017; 25:4–6.

[10] Wen HG, Zhao JH, Zhang BS, Gao F, Wu XM, Yan YS, et al. Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens. Nat Plants. 2023; 9:1409–1418.

[11] Tsatsaronis JA, Franch-Arroyo S, Resch U, Charpentier E. Extracellular vesicle RNA: a universal mediator of microbial communication? TIM. 2018; 26:401–410.

[12] Nation GK, Saffold CE, Pua HH. Secret messengers: extracellular RNA communication in the immune system. Immunol Rev. 2021; 304:62–76.

[13] Murillo OD, Thistlethwaite W, Rozowsky J, Subramanian SL, Lucero R, Shah N, et al. exRNA Atlas analysis reveals distinct extracellular RNA cargo types and their carriers present across human biofluids. Cell. 2019; 177:463–477.

[14] Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA. 2011; 108:5003–5008.

[15] Geekiyanage H, Rayatpisheh S, Wohlschlegel JA, Brown, Jr. R, Ambros V. Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides. Proc Natl Acad Sci USA. 2020; 117:24213–24223.

[16] Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011; 13:423–433.

[17] Van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018; 19:213–228.

[18] Mulcahy LA, Pink RC, Carter DRF. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014; 3:24641.

[19] Xiao D, Gao X, Xu J, Liang X, Li Q, Yao J, et al. Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle. Insect Biochem Mol Biol. 2015; 60:68–77.

[20] Ulvila J, Parikka M, Kleino A, Sormunen R, Ezekowitz RA, Kocks C, et al. Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J Biol Chem. 2006; 281:14370–14375.

[21] Saleh MC, Van Rij RP, Hekele A, Gillis A, Foley E, O’Farrell PH, et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol. 2006; 8:793–802.

[22] Fan L, Li R, Pan J, Ding Z, Lin J. Endocytosis and its regulation in plants. Trends Plant Sci. 2015; 20:388–397.

[23] He B, Wang H, Liu G, Chen A, Calvo A, Cai Q, et al. Fungal small RNAs ride in extracellular vesicles to enter plant cells through clathrin-mediated endocytosis. Nat Commun. 2023; 14:4383.

[24] Proszynski TJ, Klemm R, Bagnat M, Gaus K, Simons K. Plasma membrane polarization during mating in yeast cells. J Cell Biol. 2006; 173:861–866.

[25] Weinberg J, Drubin DG. Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol. 2012; 22:1–13.

[26] Read ND, Kalkman ER. Does endocytosis occur in fungal hyphae? Fungal Genet Biol. 2003; 39:199–203.

[27] Steinberg G. Endocytosis and early endosome motility in filamentous fungi. Curr Opin Microbiol. 2014; 20:10–18.

[28] Lee SC, Schmidtke SN, Dangott LJ, Shaw BD. Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth. Eukaryotic Cell. 2008; 7:1278–1288.

[29] Wytinck N, Sullivan DS, Biggar KT, Crisostomo L, Pelka P, Belmonte MF, et al. Clathrin mediated endocytosis is involved in the uptake of exogenous double-stranded RNA in the white mold phytopathogen Sclerotinia sclerotiorum. Sci Rep. 2020; 10:12773.

[30] Zhang T, Jin Y, Zhao JH, Gao F, Zhou BJ, Fang YY, et al. Host-Induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahliae. Mol Plant. 2016; 9:939–942.

[31] Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA, Read ND. Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J Microsc. 2000; 198:246–259.

[32] Zerial M, Mcbride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2001; 2:107–117.

[33] Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, et al. The small Gtpase Rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992; 70:715–728.

[34] Gorvel JP, Chavrier P, Zerial M, Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991; 64:915–925.

[35] Li L, Chen X, Zhang S, Yang J, Chen D, Liu M, et al. MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae. PLoS Genet. 2017; 13:e1006814.

[36] Li X, Gao C, Li L, Liu M, Yin Z, Zhang H, et al. MoEnd3 regulates appressorium formation and virulence through mediating endocytosis in rice blast fungus Magnaporthe oryzae. PLoS Pathog. 2017; 13:e1006449.

[37] Kim JH, Kim HW, Heo DH, Chang M, Baek IJ, Yun CW. FgEnd1 is a putative component of the endocytic machinery and mediates ferrichrome uptake in F. graminearum. Curr Genet. 2009; 55:593–600.

[38] Zhang T, Zhao YL, Zhao JH, Wang S, Jin Y, Chen ZQ, et al. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat Plants. 2016; 2:16153.

[39] Fuchs U, Hause G, Schuchardt I, Steinberg G. Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis. Plant Cell. 2006; 18:2066–2081.

[40] Mooren OL, Galletta BJ, Cooper JA. Roles for actin assembly in endocytosis. Annu Rev Biochem. 2012; 81:661–686.

[41] Knip M, Constantin ME, Thordal-Christensen H. Trans-kingdom cross-talk: small RNAs on the move. PLoS Genet. 2014; 10:e1004602.

[42] Chatterjee S, Kon E, Sharma P, Peer D. Endosomal escape: a bottleneck for LNP-mediated therapeutics. Proc Natl Acad Sci USA. 2024; 121:e2307800120.

[43] Witten J, Hu Y, Langer R, Anderson DG. Recent advances in nanoparticulate RNA delivery systems. Proc Natl Acad Sci USA. 2024; 121:e2307798120.

[44] Niu D, Hamby R, Sanchez JN, Cai Q, Yan Q, Jin H. RNAs—a new frontier in crop protection. Curr Opin Biotechnol. 2021; 70:204–212.

[45] Fang R. Microbe-induced gene silencing explores interspecies RNAi and opens up possibilities of crop protection. Sci China Life Sci. 2024; 67:626–628.

[46] Zhang T, Zhao JH, Fang YY, Guo HS, Jin Y. Exploring the effectiveness and durability of Trans-Kingdom silencing of fungal genes in the vascular pathogen Verticillium dahliae. Int J Mol Sci. 2022; 23:2742.

[47] Wang S, He B, Wu H, Cai Q, Ramírez-Sánchez O, Abreu-Goodger C, et al. Plant mRNAs move into a fungal pathogen via extracellular vesicles to reduce infection. Cell Host Microbe. 2024; 32:93–105.

[48] Gao F, Zhou BJ, Li GY, Jia PS, Li H, Zhao YL, et al. A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity. PLoS One. 2010; 5:e15319.

[49] Zhao YL, Zhou TT, Guo HS. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca²⁺ signaling is required for plant infection by Verticillium dahliae. PLoS Pathog. 2016; 12:e1005793.

[50] Zhou TT, Zhao YL, Guo HS. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahlias. PLoS Pathog. 2017; 13:e1006275.

[51] Wang S, Xing H, Hua C, Guo HS, Zhang J. An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption method for Verticillium dahliae. Phytopathology. 2016; 106:645–652.

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2025 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.