FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by targeting a glycosyl hydrolase coding gene at transcriptional level

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 Panama disease 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 and hypersensitivity to hydrogen peroxide (H2O2). These results indicate that milRNA biosynthesis is crucial for Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly downregulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR analysis. Through deletion and overexpression of milR-87 in the wild-type Foc strain, functions of milR-87 were studied. The results showed that milR-87 is crucial for Foc virulence in infection process. We furthermore identified a glycosyl hydrolase-coding gene, FOIG_15013, as the direct target of milR-87. The FOIG_15013 deletion mutant displayed 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. Author summary The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. As a typical representative of F. oxysporum species complex, the pathogen has been widely concerned. However, pathogenesis of Foc is not fully elucidated. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, one 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, miR-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 Panama disease.


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Banana Fusarium wilt, also known as Panama disease, is caused by the fungal pathogen In addition to the AGO coding genes, two Dicer coding genes (FoDCL1 and FoDCL2) exist in 148 Foc, which function in the biosynthesis of fungal sRNAs [11]. Compared with Foc in pure culture 149 conditions, FoQDE2 and Dicer encoding genes were significantly upregulated at 24 hours post 150 inoculation (hpi) on host banana plants. In contrast, transcription of FoAGO2 was nearly 151 undetectable ( Fig 2B). The transcriptional induction of these sRNA biosynthesis genes during host 152 infection stage suggests that the sRNAs synthesis mediated by these genes may play an active role 153 during Foc pathogenesis. 154 To further investigate the function of the three upregulated genes (FoQDE2, FoDCL1, and 155 FoDCL2), we generated these genes deletion mutants in the wild-type (WT) strain XJZ2 via  Table). 163 Relative transcript levels of FoQDE2, FoDCL1, and FoDCL2 were examined by quantitative  FoQDE2 is required for proper mycelial growth, and conidial production in Foc 170 We next assessed the biological function of FoQDE2 by examining growth and conidiation 171 phenotypes in the ΔFoQDE2 mutants. The results showed that, the colonial morphology of the 172 ΔFoQDE2 was strikingly different from that of the WT strain XJZ2. The aerial hyphae of 173 ΔFoQDE2 were much less abundant than those of the WT, and the mycelia grew close to the 174 surface of the PDA media. Such morphological change of the colonies was restored to different 175 degrees in the two complemented FoQDE2 strains (c∆FoQDE2-1 and c∆FoQDE2-2; Fig 2A). On 176 the other hand, the colony morphology was not much changed in the ΔFoDCL1 or ΔFoDCL2 177 mutants (Fig 2A).
We assessed mycelial growth rate in the WT and mutant strains. From the fourth day of 179 culture, the growth of ΔFoQDE2 was significantly slower than that of the WT (p<0.01). The growth 180 of the complemented strains was not fully recovered. The ΔFoDCL1 and ΔFoDCL2 mutants 181 displayed no difference in mycelial growth compared with the WT (S2 Table). Thus, loss of 182 FoQDE2 led to slow mycelial growth on PDA medium.

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Furthermore, microconidia production was significantly reduced in the ΔFoQDE2 mutant, 184 compared to the WT, when cultured on PDA medium for 7 days. Such conidiation defect could be 185 partially restored in the FoQDE2 complemented strains (Fig 2C). Compared with the WT,

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To elucidate the causes of the decreased conidia production in ΔFoQDE2, six reported 189 conidiation-related genes [28-31] were selected for their transcript levels assessment by qRT-PCR.

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Compared with the WT, the transcript levels of four selected conidiation-related genes, StuA, 191 FoNIIA, AbaA, and WetA, were significantly down-regulated in ∆FoQDE2. Transcription of StuA 192 and FoNIIA were restored in the FoQDE2 complemented strain (c∆FoQDE2-1), to a level 193 comparable to the WT (Fig 2D). The transcript levels of brlA and Htf1 were not affected by the loss 194 of FoQDE2 (Fig 2D). Overall, our results showed that deletion of FoQDE2 leads to morphological 195 changes, including changes in colonial morphology and reduction of mycelial growth, as well as 196 reduction in conidial production of Foc.  FoQDE2 is required for oxidative stress tolerance 215 We next tested the sensitivities of the WT and the mutant strains to oxidative stress. The 216 ∆FoQDE2 mutant was hypersensitive to 3mM H 2 O 2 when cultured on minimal medium (MM), 217 which could be restored in the complemented strains (Fig 3A and 3B). However, the ∆FoDCL1 and 218 ∆FoDCL2 mutants showed no difference in sensitivity to oxidative stress as compared to the WT 219 (Fig 3A and 3B). Taken together, FoQDE2 is required for tolerance to oxidative stress.  complemented strains did ( Fig 4A). In contrast, the ∆FoDCL1 and ∆FoDCL2 mutants caused tissue 235 necrosis similar to that of WT ( Fig 4A). Thus, FoQDE2 is required for successful invasive growth.

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Pathogenicity tests on banana (Cavendish) seedlings showed that the ΔFoQDE2 mutant was 237 unable to produce obvious vascular discoloration in the corm of banana seedling (Fig 4B and 4C) 238 and was significantly reduced in virulence on banana compared to the WT (p<0.01, Fig 4D and 4E).

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The disease index of the complemented strain (cΔFoQDE2-1) was increased but did not reach the 240 level of the WT (Fig 4E), which exhibited brown discoloration in the corm of banana seedlings. On 241 the other hand, pathogenicity of the ∆FoDCL1 and ∆FoDCL2 mutants were similar to that of WT 242 (Fig 4A-4E). These observations indicate that FoQDE2 contributes to the virulence of Foc. to 50% of vascular discoloration) and 3 (greater than 50% of vascular discoloration). were reduced in the ΔFoQDE2 mutant compared to the WT (Fig 5B). Reads starting with A or U 262 were more abundant than those starting with C or G (Fig 5C). To identify sRNA-producing loci, 263 reads with counts of 10 or higher that mapped to the UTR and intronic and intergenic regions were 264 analyzed. Overlapping and adjacent reads were grouped and only those with a consensus length of 13 265 less than 300 nt were considered to be small RNA-producing loci. Using this method, 364 loci were 266 captured in the WT and ΔFoQDE2 mutant. Among them, 25 loci were significantly decreased and 267 13 loci were significantly increased in ∆FoQDE2 compared with the WT. (Fig 5D).  The ΔmiR-87 mutant was hypersensitive to the oxidative stress generated by 3 mM H 2 O 2 , a 295 phenotype similar to that of the ΔFoQDE2 mutant ( Fig 6D). In contrast, the growth of milR-87 296 overexpress transformant (OEmilR-87-1) was not affected by oxidative stress (Fig 6D). The result 297 indicates milR-87 positively regulates tolerance to oxidative stress in Foc. 298 Pathogenicity tests showed that the ΔmiR-87 mutant was compromised in penetrating the 299 cellophane membrane and caused slighter necrosis on the surface of tomato fruits than the WT.

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While overexpressed transformant showed the opposite (Fig 6E and 6F). Infection assay using 301 banana seedlings showed that ΔmilR-87 significantly reduced in virulence compared to the WT, 302 while the virulence of overexpressed transformant was enhanced, as it caused obvious internal 303 disease symptoms of brown discoloration (Fig 6G and 6H). Furthermore, qRT-PCR analysis 304 showed that milR-87 was significantly upregulated during the early stages of infection (24, 48, and 305 96 hpi) compared with levels in pure culture (0 hpi) conditions ( Fig 6C). These results suggest that  To confirm the function of milRNA (milR-87) in Foc, synthetic double-strand siRNAs 327 (siRNA-1 and siRNA-2) and single-strand antisense small RNA (inhibitor) were designed (S1 328   Table) to silence the expression of milR-87. We observed that when the siRNAs or inhibitor was 329 transfected into Foc protoplast inoculated on the banana leaf, the size of the lesion decreased 16 330 significantly compared to those caused by the Foc protoplasts with water as control (CK) (Fig 7A   331 and 7B). An unrelated single-strand sRNA served as the negative control (NC) did not interfere 332 with lesion formation when added into the Foc protoplasts inoculated on the banana leaf ( Fig 7A   333 and 7B). We noticed that the ΔmilR-87 mutant produced the smallest lesion on the banana leaf ( Fig   334   7A and 7B). The qRT-PCR results confirmed that expression of milR-87 could be silenced by the 335 siRNA-1, siRNA-2 and inhibitor in different degree ( Fig 7C). Overall, these results confirmed that 336 milR-87 is required for full virulence of Foc.  for further investigation. The predicted targeted site of milR-87 was located in the ORF region of 361 FOIG_15013, we then generated a fusion protein by directly ligating the GFP coding sequence to 362 the C'-terminus of FOIG_15013 (Fig 8A). A point-mutated FOIG_15013 that could not be paired 363 with milR-87, FOIG_15013m, was also fused with GFP ( Fig 8A). The constructs were respectively FOIG_15013-GFP expression was not inhibited, as indicated by the strong GFP signal (Fig 8B and   368 8C). In contrast, the GFP signal was hardly detected in the WT strain, likely due to milR-87 369 mediated suppression of FOIG_15013-GFP expression (Fig 8B and 8C). The FOIG_15013m-GFP 370 expression was not affected in the WT background, as milR-87 was unable to pair with the mutation 371 site. Correspondingly, transcript level of FOIG_15013 and its mutated version in different strains 372 was consistent with that of GFP signal detection (Fig 8D), further confirming that  In order to clarify whether milR-87 regulates Foc pathogenesis through modulating 389 FOIG_15013 expression, we generated the ΔFOIG_15013 mutants (S5A and S5B Figs) and 390 characterized them in growth and pathogenicity. The results showed that, the ΔFOIG_15013 391 mutants grew faster and produced significantly more conidia than the WT strain XJZ2 when 392 cultured on PDA plate (Fig 9A and 9B). Pathogenicity tests showed that the ΔFOIG_15013 mutants 393 were more virulent than the WT (Fig 9C and 9D). These results indicate that milR-87 may promote

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After a 5-day culture on PDA plates, the colony morphology of the tested strains was recorded.

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Each experiment was repeated three times independently.

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Oxidative stress test 516 To test the sensitivity of strains to hydrogen peroxide, a conidial suspension of each strain was 517 spotted on MM or MM supplemented with H 2 O 2 (2 to 3 mM) and cultured at 28C. The colony 518 diameter was measured after 4 days' incubation. A Student's t-test was used to assess significant 519 differences between the WT and mutant.  MilRNA expression was detected using qRT-PCR, as described above, except that the reference  were identified by PCR with primers given in the table S1. Based on the sequence of milR-87, its target genes in Foc genome were predicted using 614 psRNATarget online software. In which the genes significantly up-regulated in the mutants 615 ΔmilR-87 and ΔFoQDE2 were screened by qRT-PCR and selected for target gene identification.

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According to the target site of the milRNA, a GFP marker gene was fused to the 3 'terminal of the