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FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by silencing a glycosyl hydrolase coding gene expression [1]

['Minhui Li', 'Department Of Plant Pathology', 'Guangdong Province Key Laboratory Of Microbial Signals', 'Disease Control', 'South China Agricultural University', 'Guangzhou', 'Pr China', 'Lifei Xie', 'Meng Wang', 'Yilian Lin']

Date: 2022-07 

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Banana Fusarium vascular wilt that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence, suggesting the involvement of milRNA biosynthesis in the Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly down-regulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR and Northern blot analysis. Compared to the wild-type, the deletion mutant of milR-87 was significantly reduced in virulence, while overexpression of milR-87 enhanced disease severity, confirming that milR-87 is crucial for Foc virulence in the infection process. We furthermore identified FOIG_15013 (a glycosyl hydrolase-coding gene) as the direct target of milR-87 based on the expression of FOIG_15013-GFP fusion protein. The FOIG_15013 deletion mutant displayed similar phenotypes as the overexpression of milR-87, with a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases.

The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Banana Fusarium vascular wilt that threatens global banana production. However, knowledge about pathogenesis of Foc is limited. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, an Argonaute coding gene, and two Dicer coding genes FoDCL1 and FoDCL2, which are involved in milRNA biosynthesis, are significantly induced during the early infection stage of Foc. The results suggested that the milRNAs biosynthesis mediated by these genes may play an active role in the infection process of Foc. Based on this assumption, we subsequently found a FoQDE2-dependent milRNA (milR-87) and identified its target gene. Functional analysis showed that FoQDE2, milR-87 and its target gene were involved in the pathogenicity of Foc in different degree. The studies help us gain insight into the pathogenesis with FoQDE2, milR-87, and its target gene as central axis in Foc. The identified pathogenicity-involved milRNA provides an active target for developing novel and efficient biocontrol agents against Banana Fusarium wilt.

Funding: M.L. was supported by the National Science Foundation (31800112), the Natural Science Foundation of Guangdong Province, China (2018A030313699) and the Guangdong Basic and Applied Basic Research Foundation (2020B1515420006). H.L. was supported by the China Agriculture Research System of MOF and MARA (CARS-31-09). Z.J. was supported by the Key Realm Research and Development Program of Guangdong Province (2018B020205003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

In this study, we identified an AGO and two Dicer proteins in Foc, and examined their functions in milRNAs biosynthesis and in fungal pathogenesis by sRNA sequencing and reverse genetics. We identified a FoQDE2-dependent milRNA (milR-87), which contributes to invasive growth during the initial stage of Foc infection and thus affects Foc pathogenicity, likely by targeting the gene FOIG_15013. FOIG_15013 encodes a glycosyl hydrolase, which appears as a negative regulator of Foc conidiation and pathogenicity. Overall, our findings uncover the novel function of milRNA in Foc pathogenicity.

Argonaute (AGO) proteins are evolutionarily conserved in all domains of life and play a key role in the RNA interference (RNAi) pathway [ 26 ]. As in plants and animals, AGO proteins are the core components of the RNA-induced silencing complex (RISC) and contribute to gene silencing by using the loaded sRNA as a specificity determinant in fungi [ 15 ]. QDE2, one of the two identified AGO proteins in N. crassa, functions as a slicer and is required for the biogenesis of some sRNAs such as milRNAs and PIWI-interacting RNAs [ 12 , 24 ]. Suppressor of meiotic silencing 2 (Sms-2), another reported AGO protein in the N. crassa genome, is thought to function by binding to sRNAs originating from the unpaired DNA region and is required for meiotic silencing of unpaired DNA [ 25 , 27 ]. In the model fungus N. crassa, milR-1 type of milRNAs are the most abundant milRNAs and the maturation of the milRNAs requires the AGO protein QDE-2 [ 12 , 24 ]. However, whether this type of milRNAs exist and what function they have in Foc remain unclear.

Four types of milRNAs generated from four different biosynthesis pathways, namely milR-1 to -4, have been reported in the model fungus N. crassa [ 12 ]. Different combinations of factors, including Dicers, QDE2 (Quelling Deficient 2), the exonuclease QIP (QDE2 interacting protein), and an RNAse III domain-containing protein, MRPL3, are involved in the production of milRNAs [ 12 , 23 , 24 ]. The reported milRNA biosynthesis pathways in N. crassa appear more complex and diverse than that in plants and animals [ 25 ].

MicroRNAs (miRNAs), a type of small non-coding single-stranded RNAs, play crucial roles in diverse biological processes [ 9 , 10 ]. Through base pairing with target messenger RNAs (mRNAs), miRNAs degrade the target mRNA or inhibit its translation and thereby regulate gene expression at the post transcription level [ 11 ]. Small RNAs (sRNAs) have been reported in various fungi, including Neurospora crassa, Magnaporthe oryzae, Botrytis cinerea, and Sclerotinia sclerotiorum [ 12 – 15 ]. Because some of these sRNAs are structurally similar to miRNAs from plants and animals, they are called microRNA-like RNAs (milRNAs) [ 12 , 14 ]. In B. cinerea, milRNAs have been identified as virulent effectors that suppress host immunity and facilitate fungal infection [ 14 , 16 ]. In Verticillium dahliae, a novel milRNA, VdmilR1, was reported to play crucial roles in pathogenicity [ 17 ]. Recently, a milRNA (Vm-milR37) expressed exclusively in the mycelium, was verified to contribute to pathogenicity in Valsa mali [ 18 ]. On the other hand, plants also export miRNAs or siRNAs to inhibit gene expression in fungal pathogens and confer efficient crop protection from pathogen infection [ 19 – 21 ]. Thus, the trans-kingdom sRNAs play key roles in host-pathogen interactions [ 22 ].

Banana Fusarium wilt, also known as Panama disease, is caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc). And it poses a serious threat to the banana industry worldwide [ 1 , 2 ]. Four physiological races of Foc have been identified, of which tropical race 4 (TR4) has the most devastating effect on banana production [ 2 , 3 ]. TR4 originated in Southeast Asia and is the main cause of banana Fusarium wilt in China [ 3 – 6 ]. TR4 has spread rapidly around the world, and has been reported in Mozambique, Australia, Pakistan, and even in countries along the Mediterranean coast, in countries such as Lebanon, Oman, and Jordan [ 1 , 2 , 7 ]. However, fungicides, flood fallowing, and organic amendments have rarely provided long-term control in any banana planting area. The only effective method for controlling the dissemination and subsequent infections by Foc in banana is by the quarantine or exclusion of infected properties or by planting non-host crops or cultivars [ 8 ]. Lack of effective methods to control banana fusarium wilt seriously imperils global banana production. Improved strategies to control this devastating disease are urgently needed.

Results

Identification of AGO protein FoQDE2 in Foc By performing an orthologous protein BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi) search, we identified two AGO proteins in Foc, encoded by FoQDE2 (FOIG_01986) and FoAGO2 (FOIG_01246) respectively. Conserved domain prediction showed that the proteins have four domains: a variable N-terminal domain (ArgoN), a linker 1 domain (ArgoL1), a PAZ domain, and a PIWI domain (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Phylogenetic analysis of fungal Argonaute proteins. (A) Conserved domains of Argonaute proteins were predicted through BLASTp on NCBI. (B) Fungal Argonaute protein sequences were first aligned using Clustal×1.8 and then the aligned sequences were analyzed by the Maximum Likelihood method implemented in MEGA 7. Bootstrap values are expressed as a percentage of 1000 replicates. In addition to Argonaute proteins from the Fusarium oxysporum f. sp. cubense (Foc) genome showed in bold, proteins used for this analysis include Agl1–Agl4 from Cryphonectria parasitica [28], a chestnut blight fungus; MoAGO1–MoAGO3 from Magnaporthe oryzae, a rice blast fungus [15]; NcQDE2 and NcSMS-2 from the model fungus Neurospora crassa [25]; and proteins from the F. graminearum (FGSG), F. verticillioides (FVEG), and F. oxysporum (FOVG: GCA_000260075.2; FOQG: GCA_000260235.2; FOTG: GCA_000260175.2; FOMG: GCA_000260495.2; FOXG: GCA_000149955.2; and FOYG: GCA_000271745.2) genomes. https://doi.org/10.1371/journal.ppat.1010157.g001 The amino acid sequences of AGO orthologs in other fungal species were retrieved from GenBank for phylogenetic analysis. Two to five AGO-like proteins were identified in different strains of F. oxysporum, but only two in Foc. Phylogenetic analysis indicated that AGO proteins from filamentous fungi could be divided into three subgroups (Fig 1B). In the first group, FoQDE2 and its orthologs from F. oxysporum and F. graminearum were clustered with QDE2 from N. crassa, AGO-like protein MoAGO3 from M. oryzae [15], and Agl1 from the chestnut blight fungus Cryphonectria parasitica [28]. The second group includes AGO-like proteins from F. oxysporum and Agl2 and Agl3 from C. parasitica. The third group is composed of FoAGO2 and its orthologs from F. oxysporum and F. graminearum, Agl4 from C. parasitica, MoAGO1 from M. oryzae, and SMS2 from N. crassa [25]. The phylogenetic analysis showed that the AGO proteins in Foc are orthologous to those of the other filamentous fungi.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010157

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