# Plant-parasitic nematode microRNAs hijack plant AGO1 to induce host-cell reprogramming

Ange Dussutour, Yara Noureddine, Martine da Rocha, Oussama Yahmi, Abile T. Mohammed, Karine Mulet, Pauline Foubert, Ercan Seçkin, An-Po Cheng, Jérôme Zervudacki, et al.

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Ange Dussutour, Yara Noureddine, Martine da Rocha, Oussama Yahmi, Abile T. Mohammed, et al. Plant-parasitic nematode microRNAs hijack plant AGO1 to induce host-cell reprogramming. 2026. [hal-05513624](https://hal.inrae.fr/hal-05513624v1)

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1 Plant-parasitic nematode microRNAs hijack plant AGO1 to induce host-cell

2 reprogramming

3 Ange Dussutour¹, Yara Noureddine¹, Martine da Rocha¹, Oussama Yahmi¹, Abile T.

4 Mohammed¹, Karine Mulet¹, Pauline Foubert¹, Ercan Seçkin¹, An-Po Cheng², Jérôme

5 Zervudacki³, Lionel Navarro³, Arne Weiberg², Michaël Quentin¹, Bruno Favery¹ and

6 Stéphanie Jaubert¹*

7

8

9

10

11 1. INRAE, Université Côte d'Azur, ISA, 06903 Sophia Antipolis, France

12 2. Universität Hamburg, Fakultät für Mathematik, Informatik und Naturwissenschaften

13 Fachbereich Biologie, Institut für Pflanzenwissenschaften und Mikrobiologie

14 RNA Infektionsbiologie der Pflanze, Ohnhorststr. 18, 22609 Hamburg

15 3. Institut de Biologie de l'ENS (IBENS), Ecole Normale Supérieure, Université PSL, CNRS,

16 INSERM, F-75005, Paris, France.

17

18 * corresponding author

19

20 ABSTRACT

21 Cross-kingdom RNA interference (ckRNAi) is emerging as a mode of inter-organismal gene
22 regulation, yet mechanistic examples in plant–metazoan interactions remain limited. Here, we
23 demonstrate miRNA-driven ckRNAi in the nematode–plant pathosystem. Root-knot
24 nematodes are among the most destructive plant pathogens, reprogramming root tissues to
25 develop into galls containing multinucleated, hypermetabolic giant feeding cells essential for
26 parasitism. AGO1-associated small-RNA immunoprecipitation (AGO1-RIP) from tomato
27 galls revealed the selective in planta loading of 10 M. incognita miRNAs into host AGO1.

28 Integrating degradome profiling, target prediction, and dual-luciferase reporter assays, we
29 validated miRNA-directed silencing of nine tomato transcripts by four secreted nematode
30 miRNAs. These targets map to major pathway classes involved in immune signaling,
31 metabolic regulation, and cellular reprogramming linked to feeding-site establishment.

32 Functional analyses further show that the nematode-secreted miR-2b is enhances giant
33 feeding cell development. Comparative AGO1-RIP in Arabidopsis thaliana identified a
34 conserved subset of AGO1-loaded nematode miRNAs, including miR-2b and miR-100,
35 consistent with shared small-RNA effectors across hosts. Finally, the overlap between AGO1-
36 loaded miRNA families and helminth secreted small-RNA repertoires supports evolutionary
37 convergence on RNA-based virulence strategies. Collectively, our findings establish miRNA-
38 mediated ckRNAi as a mechanistic component of plant–root-knot nematode interactions and
39 provide a framework for leveraging RNA-based vulnerabilities for nematode control.

40

41

INTRODUCTION

42 RNA interference (RNAi) is a highly conserved mechanism of control over gene expression
43 through small non-coding RNAs (sRNAs) (Vaucheret et al., 2004; Bartel, 2009; Bartel, 2018).
44 These sRNAs are typically 18-25 nucleotides (nt) long and are incorporated into Argonaute
45 (AGO)-containing RNA-induced silencing complexes (RISC), which recognize
46 complementary messenger RNAs (mRNAs) by base-pairing and direct posttranscriptional
47 gene silencing (PTGS) by endonucleolytic cleavage and/or translational repression (Ameres
48 and Zamore, 2013). MicroRNAs (miRNAs) constitute a major class of sRNAs in plants. They
49 frequently act through extensive complementarity to guide the silencing of target mRNAs,
50 thereby shaping development, stress adaptation, and immune regulation.

51 Beyond their canonical intracellular functions, sRNAs have emerged as mobile signals that
52 can act on an organism other than the one in which they were produced, during interactions
53 between these two organisms. Over the last decade, evidence has accumulated that pathogens
54 and symbionts can transfer sRNAs into host cells to reprogram gene expression and promote
55 colonization, persistence, or mutualistic outcomes (Weiberg et al., 2013; Buck et al., 2014).
56 This interorganismal regulation — commonly referred to as cross-kingdom RNA interference
57 (ckRNAi) — has been documented in diverse biological systems (Cai et al., 2019; Qiao et al.,
58 2023). In plants, several fungi, oomycetes, and bacteria have been reported to deploy sRNAs
59 that engage host AGO proteins and suppress host immunity, establishing sRNAs as a distinct
60 class of virulence signals alongside proteins and metabolites (Wong-Bajracharya et al., 2022;
61 Chowdhury et al., 2024; Zhang et al., 2024). Models of the mechanisms underlying sRNA
62 exchange are continually being refined, with the incorporation of roles for extracellular RNA
63 carriers and vesicle-associated pathways, but two prerequisites for functional ckRNAi have
64 consistently been identified: sRNAs must access the appropriate host silencing machinery and
must exert sequence-specific regulatory effects on host transcripts. The demonstration of functional engagement is crucial, because non-host sRNAs may be detected in infected tissues due to extracellular contamination or extraction artifacts.

Metazoan-plant ckRNAi has been characterized in much less detail than plant-microbe systems. A few studies have provided evidence that herbivorous insects can deliver regulatory sRNAs that modulate plant gene expression, but the extent to which this phenomenon is generalized among metazoan plant pathogens remains unclear (Zhang et al., 2024; Han et al., 2025). This constitutes a major gap in our knowledge, given the considerable reliance of several metazoan parasites on intimate, long-term interactions with plant tissues, potentially involving RNA-based effector strategies. Determining whether metazoan plant pathogens deploy sRNAs as bona fide effectors — i.e., sRNAs that enter host cells, load host AGO complexes and direct the silencing of host targets — would therefore advance our fundamental understanding of host manipulation, potentially expanding the conceptual framework for RNA-based disease control.

Root-knot nematodes (RKNs) of the genus *Meloidogyne* are highly adapted, economically important plant parasites that rank among the most damaging plant pathogens worldwide. *Meloidogyne incognita* is particularly notable for its exceptionally broad host range, infecting more than 5,000 plant species, including most cultivated vegetable crops (Castagnone-Sereno, 2006; Blok et al., 2008; Abad and Williamson, 2010). Despite this diversity of hosts, RKNs reproducibly induce morphologically and functionally similar feeding sites, highlighting their capacity to exploit cellular programs that are conserved in plants. After invading the roots, juveniles establish a permanent feeding site by reprogramming a small number of vascular parenchyma cells into multinucleated, highly metabolically active giant cells that function as strong nutrient sinks required for nematode growth and reproduction. The formation and maintenance of these feeding structures requires extensive remodeling of host transcription and physiology in response to nematode-derived signals (Favery et al., 2020). The esophageal glands of RKNs produce a large repertoire of secreted effectors, which are delivered via the stylet, making it possible to manipulate plant immunity, cell cycle control, development, and RNA metabolism directly (Mejias et al., 2019; Mejias et al., 2022; Rutter et al., 2022). Protein effectors play a well-established role as central drivers of parasitism, but the extent to which sRNAs contribute to nematode-induced host reprogramming remains unclear. Plant-nematode interfaces are already known to allow biologically meaningful RNA transfer in the opposite
direction: host-induced gene silencing (HIGS), in which plants express dsRNA/siRNAs
targeting nematode genes, can reduce nematode parasitism (Gheysen and Vanholme, 2007).

In this context, miRNA-based ckRNAi is a plausible and potentially powerful strategy. Many core regulatory and signaling nodes are conserved across plant species and miRNA effectors targeting these nodes may explain how a single nematode species can manipulate host pathways common to diverse plants. Moreover, the functional engagement of nematode miRNAs by the plant silencing machinery would provide a direct route for the suppression of host defenses or fine-tuning of developmental and metabolic programs associated with feeding-site formation. However, stringent evidence is required to support claims of ckRNAi in complex infected tissues, as galls contain a mixture of plant and nematode material and RNA profiles can be confounded by tissue admixture or non-specific associations during extraction. Approaches in which functional loading into host AGO proteins is used as the readout, rather than the bulk detection of parasite RNAs, therefore provide a stringent framework for distinguishing between ckRNAi candidates and background.

We investigated the engagement of *M. incognita*-derived miRNAs by the plant AGO1 pathway during gall development. We used AGO1-associated sRNA immunoprecipitation (AGO1-RIP) from infected tomato roots as a functional readout for the sRNAs present in the host RISC, coupled with stringent controls to identify nematode sRNAs potentially displaying non-specific association with AGO1 during sample processing. We then used degradome evidence *and in planta* reporter-based assays to investigate whether candidate nematode miRNAs directed the sequence-specific cleavage of host transcripts. By performing AGO1-associated profiling on phylogenetically distant host plants, we investigated whether a subset of nematode miRNAs was widely deployed across hosts and whether their predicted/validated target engagement was consistent with the modulation of conserved pathways linked to feeding-site formation. Finally, functional analyses further establish nematode-secreted miR-2b as a determinant of giant feeding cell formation. Together, these findings establish a functional framework for evaluating miRNA-based ckRNAi in plant–nematode interactions and clarifying the role of nematode miRNAs as effectors in the molecular dialog underlying RKN parasitism.

126

127
128

129 **RESULTS**

130 **Genome-wide identification of *Meloidogyne incognita* miRNAs**

131 We characterized the *M. incognita* sRNA repertoire during parasitism by generating sRNA
132 libraries from freshly hatched, preparasitic second-stage juveniles (J2) and from parasitic
133 stages embedded in tomato galls at 7 and 14 days post infection (dpi). High-quality reads
134 were aligned with *M. incognita* genome assembly V3 (Blanc-Mathieu et al., 2017) and used
135 for *de novo* miRNA annotation.

136 By integrating miRDeep2 and ShortStack predictions and curating conserved loci, we
137 identified 288 MIR genes in the M. incognita V3 genome, including 119 miRNAs with
138 orthologs in miRbase. These 288 MIR genes were grouped into 158 miRNA families
139 (Supplemental Figure 1 and Supplemental Table 1) and organized into 17 genomic clusters
140 (Supplemental Table 2). A substantial proportion of these MIR loci corresponded to putative
141 lineage-specific miRNAs (i.e., with no detectable homologs in available nematode or plant
142 genome sequences; number = 167). A core set of 15 conserved miRNAs (miR-100, miR-1,
143 miR-71, lin-4, miR-5359, miR-92, miR-124, miR-50, miR-86, miR-87, miR-7904, miR-2a,
144 miR-2b, miR-279, miR-252; ranked by abundance), and one Meloidogyne-specific miRNA
145 (miR-minc70) dominated the sRNA pool (Figure 1; Supplemental Table 3). A small number
146 of conserved miRNA families therefore account for a large proportion of total miRNA reads
147 despite extensive lineage-specific diversification.

148

149 **M. incognita miRNAs hijack host AGO1 during infection**

150 RKN are obligatory sedentary endoparasites, which makes it difficult to isolate the secretion
151 they produce *in planta*. We investigated whether *M. incognita* sRNAs were delivered to plant
152 cells and engaged the host RISC, by performing AGO1 RNA immunoprecipitation (AGO1-
153 RIP) on tomato (*Solanum lycopersicum*) root galls at 14 dpi with anti-AGO1 antibodies
154 previously validated for plant AGO1-RIP assays (Dunker et al., 2021). Galls are a mix of
155 plant and nematode tissues. We therefore used a stringent negative control in which
156 uninfected tomato roots were mixed with preparasitic J2s immediately before tissue
disruption, thereby enabling to identify nematode sRNAs that might bind AGO1 non-
specifically during extraction.

We removed ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) reads, retaining only unique sRNA sequences present at a frequency of >50 reads per million (rpm) in at least two biological replicates. We identified 236 unique sRNA sequences associated with SlAGO1 displaying the expected enrichment in 22-nt species with a 5' uracil residue (Figure 1, Supplemental Table 4). The mapping of AGO1-associated sRNAs onto the tomato (ITAG 3.2) and *M. incognita* (V3) genome sequences resulted in 218 sequences being assigned to tomato and 18 to *M. incognita*.

Eleven of the 18 nematode-derived AGO1-associated sRNAs were annotated *M. incognita*
miRNAs (Table 1). These 11 nematode miRNAs accounted for 19.6% of all AGO1-RIP reads
in infected galls, corresponding to an enrichment of about 50-fold relative to the negative
control, in which nematode sRNAs accounted for 0.4% of AGO1-associated reads. The set of
AGO1-associated nematode miRNAs consisted of nine conserved miRNAs (miR-2b, miR-
5359, miR-100, miR-92, miR-58, miR-71, let-7, miR-7904, miR-57; ordered by abundance)
and two *Meloidogyne*-specific miRNAs (miR-Miex1 and miR-Miex2).

Three nematode microRNAs — miR-2b, miR-5359 and let-7 — were also found in the control. An analysis of differential expression between galls and the negative control revealed a massive enrichment in miR-2b and miR-5359 in galls, whereas the abundance of let-7 was similar in galls and the control (Supplemental Table 5). This result suggests that the association of let-7 with SlAGO1 may be due to non-specific association during the grinding together of plant and nematode tissues. We therefore excluded let-7 from further functional analysis. Overall, these results demonstrate a strong and specific enrichment of a defined set of *M. incognita* miRNAs in the SlAGO1 complex during infection, supporting their active delivery in host cells.

The selective enrichment of AGO1-associated nematode miRNAs does not correlate to nematode miRNA abundance

We then investigated whether the abundances of nematode miRNAs in SlAGO1-RIP reflected
their overall abundance in the nematode. In tomato gall AGO1-RIP datasets, miR-2b was the
most abundant nematode miRNA species — at a median of 622 rpm among nematode-
188 associated reads and with a mean accumulation of ~43,000 rpm overall — accounting for
189 14.7% of all AGO1-associated sRNAs and constituting the dominant component of the
190 nematode-derived fraction. At whole-library level, miR-2b was the second most abundant
191 AGO1-associated sRNA species, surprisingly exceeded only by the sly-miR-482b miRNA of
192 the host plant, tomato (Supplemental Table 4).

193 We then assessed the abundance of *M. incognita* miRNA in sRNA-seq datasets derived from
194 preparasitic J2s (Figure 1 and Supplemental table 3). Several nematode miRNAs detected in
195 SIAGO1-RIP (miR-100, miR-71, miR-5359, miR-92, miR-7904, and miR-2b) were among
196 the top 15 most abundant miRNAs in preparasitic stages. By contrast, other SIAGO1-
197 associated miRNAs (miR-Miex1, miR-Miex2, miR-58, miR-57) were not among the most
198 abundant miRNAs in nematode bulk datasets. Conversely, several nematode miRNAs found
199 to be highly expressed in J2/G14 datasets (miR-1, miR-124, lin-4, miR-50, miR-87, miR-279)
200 were absent from SIAGO1-RIP libraries. The ranking of AGO1-associated nematode
201 miRNAs by abundance therefore displayed limited concordance with the overall abundance of
202 these molecules in nematode sRNA-seq datasets. An extreme example was provided by miR-
203 2b, which had a low abundance in nematode bulk datasets but was very frequent among
204 AGO1-associated reads *in planta*. Together, these results indicate that the nematode sRNAs
205 delivered to plant cells and loaded on plant AGO1 constitute a selective subset of nematode
206 miRNAs rather than a passive reflection of the overall nematode miRNA pool.

207 Identification of the targets in tomato of the microRNAs secreted by *M. incognita*

208 For the identification of putative tomato transcripts targeted by AGO1-associated nematode
209 miRNAs, we combined degradome analyses with computational target prediction. We first
210 reanalyzed a tomato 14 dpi gall degradome library (Noureddine et al., 2023) with Cleaveland4
211 and the 10 nematode miRNAs retained after the exclusion of let-7. Using ≤2 as the
212 degradome category cutoff, we identified 63 candidate tomato targets of the 10 nematode
213 miRNAs (Supplemental Table 6). Target numbers were heterogeneously distributed between
214 miRNAs: miR-2b, miR-5359, miR-92, and miR-Miex1 were associated with 43 targets, with
215 miR-5359 associated with the largest number of targets for any single miRNA (22 targets). By
216 contrast, miR-58, miR-Miex2, miR-7904, miR-57, and miR-100 together accounted for only
217 13 targets, and no degradome-supported target was recovered for miR-71 with the application
218 of these criteria.
219 Degradome sensitivity can be reduced by tissue complexity, nematode sRNA dilution and the
220 rapid degradation of cleavage products. We therefore performed complementary *in silico*
221 prediction with psRNATarget. This analysis identified 201 putative targets (mean of ~20
222 targets per miRNA; Supplemental Table 7). Consistent with the degradome analysis, the
223 number of predicted targets was highest for miR-5359 (57) and lowest for miR-57 (2).
224 Relative to degradome-based results, psRNATarget predicted larger target sets for miR-7904,
225 miR-58, miR-Miex2, and miR-71 (25-31 predictions per miRNA). Nineteen transcripts were
226 predicted as target by both analyses for eight of the secreted nematode miRNAs. 29 candidate
227 targets were then selected for further experimental testing of silencing *in planta*
228 (Supplemental Table 8).

229 *In planta* validation of nematode miRNA silencing activity with dual-luciferase reporters

230 We investigated the ability of nematode miRNAs to silence predicted tomato targets in plant
231 cells, in an *in planta* dual-luciferase reporter (DLR) assay (Liu et al., 2014) and by the
232 expression of artificial miRNAs (Schwab et al., 2010). For each nematode miRNA, we
233 included a positive silencing control reporter bearing a perfectly complementary target site to
234 ensure expression of the nematode miRNA *in planta* and its efficient silencing. Empty vector
235 was used as a negative cleavage control. The R-luc/F-luc ratios of targets fused to the F-luc
236 3'UTR and co-expressed with the miRNAs were normalized against the basal R-luc/F-luc
237 ratio of the negative control.

238 For the nine secreted nematode miRNAs tested, silencing activity was confirmed for miR-2b,
239 miR-5359, miR-92, miR-57, miR-58, miR-Miex1, and miR-7904, which yielded significant
240 decreases in reporter activity for the perfect-complementarity control (Supplemental Figure
241 2). Silencing strength varied between the miRNAs, with miR-2b and miR-7904 decreasing the
242 reporter activity of the positive control relative to the negative control by ~65%, whereas
243 miR-92, miR-Miex1, miR-57, and miR-58 yielded more moderate decreases in reporter
244 activity (~20-45%). The strongest activity was observed for miR-5359, which decreased the
245 F-luc signal of the positive control by >97%. By contrast, no decrease in reporter activity was
246 observed for miR-100 or miR-Miex2 with either of the positive control constructs. These
247 miRNAs were therefore either not expressed or not active in this assay configuration under
248 our experimental conditions.
249 Nine of the 29 candidate targets in tomato tested displayed significant decreases in F-luc activity following co-expression with the corresponding nematode miRNA. We identified 250 three targets for miR-2b (*Solyc06g067890*, *Solyc09g091540*, *Solyc12g049320*), two for miR-251 252 5359 (*Solyc11g006760*, *Solyc11g045110*), two for miR-Miex1 (*Solyc06g072690*, 253 *Solyc07g065890*), and two for miR-7904 (*Solyc03g118010*, *Solyc11g005450*) (Figure 2). 254 Thus, four of the nine miRNAs tested silenced nine of the 29 targets *in vivo* (~31%), 255 providing evidence in support of the functional targeting of specific tomato transcripts by 256 plant AGO1-associated nematode miRNAs (Table 2).

257 **Comparative AGO1-RIP reveals a conserved subset of nematode miRNAs in host plants**

258 Given the broad host range of *M. incognita*, we investigated the possible conservation of the 259 AGO1-associated nematode miRNA repertoire between evolutionarily distant hosts. We 260 performed AGO1-RIP on *Arabidopsis thaliana* galls at 14 dpi and profiled AGO1-associated 261 sRNAs. This analysis detected miR-2b, miR-100, let-7 and miR-57 as nematode-derived 262 miRNAs present in AGO1-associated sRNA pools in *Arabidopsis*, as they are in tomato 263 (Supplemental Table 9), indicating that at least a subset of AGO1-associated *M. incognita* 264 miRNAs are common to divergent host species.

265 By contrast to the results obtained for miR-2b, we were unable to validate the silencing of the 266 tested putative targets of miR-57 *in planta* and no silencing activity could be assessed for 267 miR-100 in our experimental conditions. We then further assessed conservation of validated 268 target sites of miR-2b for which orthologs were available. The *Arabidopsis* ortholog of the 269 tomato miR-2b target SIMT2 *SALICYLIC ACID METHYLTRANSFERASE-2* 270 (*Solyc09g091540*), *JASMONIC ACID CARBOXYL METHYLTRANSFERASE (AtJMT)* 271 (*At1g19640*), retained a conserved target site (Supplemental Figure 3). By contrast, orthologs 272 corresponding to other tomato miR-2b targets displayed sequence divergences that decreased 273 the near-perfect complementarity at the predicted target sites, consistent with potential host- 274 dependent differences in cleavage compatibility. Conservation of the miR-2b target site across 275 orthologs points to a conserved miRNA-controlled pathway that likely underpins a core M. 276 incognita infection strategy across its broad host range.

277 **Conservation and genomic organization of secreted miRNAs in different Meloidogyne**
278 **species**
279 In the *M. incognita* genome, the miRNAs released into the host belonged to 10 genomic clusters (Supplemental Table 2). Clustered miRNAs are transcribed as a single primary transcript (pri-miRNA), which is then processed into multiple precursor and mature miRNAs. 280 281
282 Several of the loci encoding miRNA released into the host (including copies of miR-2b, miR-71, and miR-5359) clustered with *MIR* genes encoding miRNAs not detected in AGO1-RIP datasets. 283 284

285 We evaluated the conservation of secreted miRNAs across RKNs, by performing 286 microsynteny analyses to identify precursor (pre-miRNA) orthologs in three additional 287 *Meloidogyne* genomes (*M. enterolobii*, *M. arenaria*, and *M. javanica*) (Supplemental Table 288 10). We were able to identify orthologous precursor sequences for most of the secreted 289 miRNAs, generally with similar copy numbers across species consistent with reported ploidy 290 differences. For many *MIR loci*, the surrounding genomic context was also conserved, 291 supporting orthology at locus level. 292

292 The miR-2 family in *Meloidogyne*: copy number, variants, and secretion-associated patterns 293

294 The most abundant nematode-derived miRNA associated with host AGO1 was miR-2b, 295 accounting for 86% of all reads assigned to secreted nematode miRNAs (Table 1). We 296 investigated the genomic basis of this enrichment in *Meloidogyne*. In *M. incognita*, we 297 identified eight *MIR-2* loci encoding three mature variants: the AGO1-associated miR-2b and 298 two variants not detectably associated with host AGO1 under our conditions (miR-2a and 299 miR-2c). These variants had identical seed sequences but differed outside the seed region 300 (Supplementary Figure 4).

301 Small-RNA profiling across developmental stages revealed broadly similar expression 302 patterns for miR-2 variants in preparasitic J2s (Supplemental Table 3). However, despite the 303 detectable expression of multiple miR-2 variants, only miR-2b displayed strong and 304 reproducible enrichment in plant AGO1-RIP libraries, consistent with selective association 305 with the host silencing machinery.

306 Consistent with our observations in *M. incognita*, miR-2 family members have been detected 307 in the secreted/extracellular fractions of several animal-parasitic nematodes (Sotillo et al., 308 2020; Chowdhury et al., 2024), indicating that host exposure to miR-2 is observed across
309 phylogenetically distant parasitic systems. We then assessed the genomic organization of
310 miR-2 loci. In many metazoans, *MIR2* genes occur in family-specific clusters, consistent with
311 tandem duplication. By contrast, in nematodes, miR-2 loci are frequently found as singletons
312 or within heterogeneous miRNA clusters. Consistent with this pattern, *M. incognita* *MIR2*
313 genes were distributed heterogeneously between singletons and heterogeneous clusters, rather
314 than forming a single family-specific cluster (Supplemental Figure 4). The extension of this
315 analysis to additional nematode genomes revealed substantial variation in miR-2 copy number
316 (range: 3 to 11 loci; Supplemental Figure 4). With eight loci, *M. incognita* was one of the
317 species with the highest miR-2 copy number in our dataset, together with *A. suum* (11 loci)
318 and *B. malayi* (10 loci). In species encoding multiple mature miR-2 variants, only a subset of
319 these variants were detected in host-exposed/AGO1-associated datasets, consistent with the
320 variant-specific enrichment observed for miR-2 family members during parasitism.

321 **miR-2b Promotes Feeding Cell Expansion**

322 We investigated whether loading on the host AGO1 reflected a function in parasitism by
323 generating tomato roots overexpressing either miR-2b or miR-57 using *Agrobacterium*
324 *rhizogenes* mediated transformation system. We investigated the effect of overexpressing
325 secreted nematode miRNAs within these transformed roots on the formation of feeding cells
326 by measuring the size of the nematode-induced feeding cells within these roots. Tomato roots
327 transformed with an empty vector were used as a negative control. The overexpression of
328 miR-2b in tomato roots induced a clear increase in feeding site size, whereas no effect was
329 observed for miR-57 (Figure 3). Altogether, these findings support a role for miR-2b in
330 promoting feeding cell formation and/or sustaining their maintenance during infection.

331

332 **DISCUSSION**

333 Our data support a model in which M. incognita deploys a highly selective, miRNA-based
334 ckRNAi strategy: a restricted set of nematode miRNAs enters host root cells, loads into plant
335 AGO1, and hack plant silencing pathway. Functional validation indicates that these miRNAs
336 converge on key host hubs controlling immunity, metabolism, and cell fate, consistent with a
337 coordinated reprogramming of gall development and feeding-site function during infection.

338 **Selective sorting and AGO1 loading of nematode miRNAs in host cells**
339 CkRNAi is based on the selective transfer of a defined subset of sRNAs into interacting
340 organisms, in which these molecules engage the RNAi machinery and mediate the silencing
341 of target genes (Weiberg et al., 2013; Dunker et al., 2020). Our results are consistent with the
342 effective selection of certain miRNAs for secretion into host cells. The nematode miRNAs
343 associated with the plant AGO1 are not simply the miRNAs most abundant in the nematode.
344 Several highly expressed nematode miRNAs are absent from the host AGO1, whereas some
345 of the miRNAs associated with AGO1 are not among the predominant species in the
346 nematode. This lack of coupling strongly argues that there is selective sorting and/or selective
347 stabilization of a restricted subset of miRNAs for delivery in the host. Moreover, the
348 pronounced enrichment (30-fold) in *M. incognita* miRNAs relative to the negative control
349 displayed by the host AGO1 demonstrates not only the secretion and delivery of these
350 miRNAs into host cells but also their functional integration into the central effector of the
351 plant RNA silencing pathway. Finally, some miRNAs destined for secretion that are encoded
352 by genes clustered with non-secreted miRNA genes within the nematode genome were found
353 to be secreted and loaded into the host AGO1. This organization may suggest that the
354 clustered miRNAs are transcribed in the same cells, implying that delivery to the host is
355 unlikely to be solely due to expression in a specific cell type. However, differential
356 maturation, the stability of mature miRNAs, and posttranscriptional regulation may result in
357 imbalances between the expression profiles of members of the same cluster (Martinez et al.,
358 2008; Jovelin and Cutter, 2014).

359 **Nematode-secreted miRNAs actively cleave specific tomato transcripts**

360 By combining degradome profiling, *in silico* target prediction, and in planta DLR assays, we
361 identified secreted nematode miRNAs capable of cleaving specific tomato transcripts.
362 Because galls comprise only a few giant feeding cells embedded within proliferating
363 surrounding tissues, cleavage events are expected to occur in a very limited cell population,
364 thereby diluting the signal in whole-gall degradome datasets and likely accounting for the low
365 degradome categories observed for candidate targets. DLR assays revealed modest but
366 reproducible repression (typically ~10–20%), lower than in some reported plant–pathogen
367 systems (Meng et al., 2021; Mei et al., 2025), and variable across miRNA–target pairs. The
368 absence of activity for miR-100 and miR-Miex2 in our positive control context may reflect
369 technical constraints—such as inefficient amiRNA processing or expression, or suboptimal
370 AGO1 loading—rather than a lack of intrinsic targeting potential.
371 Conservation across hosts and implications for host range

372 *M. incognita* miR-2b, miR-57 and miR-100 were found to be loaded into AGO1 in galls from
373 *S. lycopersicum* and *A. thaliana*, demonstrating the secretion of a small set of conserved *M.
374 incognita* miRNAs in phylogenetically distant host species and suggesting that these miRNAs
375 may be factors involved in the manipulation of conserved plant mechanisms during RKN
376 infection. As in tomato, miR-2b is one of the main miRNAs secreted in *Arabidopsis* galls,
377 supporting the hypothesis that miR-2b plays an important role in *Meloidogyne* infection
378 processes. Nevertheless, the ubiquitous secretion of large amounts of miR-2b and the low
379 level of cleavage site variation between plant jasmonate methyltransferase homologs support
380 the hypothesis that the JA hormonal defense pathway is a major and conserved target in
381 diverse plants within the broad host range of *M. incognita*. Target sequence divergence
382 probably also restricts some silencing activities to specific hosts, potentially contributing to
383 quantitative differences in susceptibility.

384 Host pathways targeted by secreted miRNAs

385 Nine secreted nematode miRNA–tomato gene pairs were validated by DLR assays. The
386 targets of the four active miRNAs (miR-2b, miR-7904, miR-Miex1, and miR-5359) belong to
387 a restricted set of pathways playing key roles in plant-RKN interactions, including host
388 defense processes commonly targeted by ckRNAi in other plant-pathogen systems (Weiberg
389 et al., 2015; Cai et al., 2019) and metabolic and developmental programs particularly relevant
390 to RKN parasitism (Favery et al., 2020). Hormone-mediated immunity (SA/JA/ET) emerged
391 as a prominent node, as silencing was confirmed for SIMT2 (*Solyc09g091540*; targeted by
392 miR-2b), SIMED26c encoding a Mediator complex subunit (*Solyc11g005450*; targeted by
393 miR-7904), the uridine kinase UKL3 (*Solyc07g065890*; targeted by miR-Miex1), and
394 possibly a GRAS transcription factor (*Solyc12g049320*); together, these findings are
395 consistent with an attenuation of defense signaling during infection (Wang et al., 2019; Kong
396 et al., 2020; Cheng et al., 2025). Other secreted miRNA target pathways linked to feeding-site
397 establishment and maintenance, notably energy metabolism and cellular reorganization, were
398 also identified: TSN (*TUDOR-SN nuclease*, *Solyc03g118010*) and SPS4F (a sucrose-
399 phosphate synthase, *Solyc11g045110*), targeted by miR-7904 and miR-5359 respectively, are
400 linked to SNF1-related protein kinase (SnRK1)-dependent metabolic control, a central
401 regulatory hub and master metabolic regulator (Nukarinen et al., 2016; Van Leene et al.,
402 2022). Furthermore, miR-2b and miR-5359 also silence transcripts encoding an alpha/beta
403 hydrolase (*Solyc06g067890*) and a multiple myeloma tumor-associated protein 2
404 (Solyc11g006760), the homologs of which in *Arabidopsis* AT1g80280 (XOPAM-ACTIVATED RESISTANCE 1, AMAR1) and AT3G52220 (a LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTOR (LILR)-like domain protein) are linked to immune-related functions (Xie *et al.* 2023). Beyond direct defense and metabolic outputs, silencing of the GRAS transcription factor, MED26c, and TSN would be expected to have broader effects on transcriptional and posttranscriptional regulation, given the reported roles of these molecules in symbiotic programs, RNA polymerase II-mediated transcriptional reprogramming, and the RISC-associated control of endogenous miRNA abundance (Jaiswal *et al.*, 2022; Freytes *et al.*, 2024; Jiang *et al.*, 2025). Finally, the targeting of the tomato *GAMMA-TUBULIN COMPLEX COMPONENT 3* (GCP3) homolog (*Solyc06g072690*) by miR-ex1 suggests a role in regulation of cytoskeletal organization and cell division required for formation of in giant feeding cells (Caillaud *et al.*, 2008; Banora *et al.*, 2011). Together, these results support a model in which nematode-secreted miRNAs orchestrate a coordinated suite of regulatory effects that damp down host immunity while directing changes in metabolism and cellular architecture to promote giant-cell development and sustain feeding-site function throughout infection.

420 **miR-2 family expansion and subfunctionalization**

421 The exceptional accumulation of miR-2b within AGO1 complexes, accounting for a 422 substantial fraction of total AGO1-associated sRNAs, identifies this miRNA as a major cross-423 kingdom silencing effector operating during infection. The miR-2 family is an ideal 424 framework in which to investigate how miRNA gene expansion can be harnessed for 425 parasitism. *Meloidogyne* species have multiple miR-2 loci encoding different mature variants 426 (miR-2a/2b/2c) with identical seeds, but with only one variant (miR-2b) displaying consistent 427 enrichment among host AGO1-bound sRNAs and ranking among the most abundant 428 nematode-derived sRNAs in infected tissues. This marked asymmetry fits a 429 subfunctionalization model, with miR-2 paralogs partitioning roles between endogenous 430 regulation and cross-kingdom delivery, mirroring patterns described for duplicated protein-431 coding genes (Blanc Matthieu *et al.* 2017). the context of the plant RNAi machinery, seed 432 identity alone is insufficient to predict functional equivalence, as base-pairing outside the seed 433 contributes to target recognition in plants, in which near-perfect complementarity is a major 434 determinant of silencing activity (Liu *et al.*, 2014). Accordingly, modest sequence differences 435 between miR-2 variants might dramatically alter the availability of quasi-complementary 436 target sites in host transcripts, while simultaneously affecting secretion-associated sorting.
437 Notably, the poor correlation between abundance and association with the host AGO1
438 observed for nematode miR-2b indicates that secretion is not driven solely by expression
439 level, providing additional support for the existence of selective trafficking mechanisms.

440 Evolutionary convergence of secreted miRNAs in parasitic nematodes

441 The selective enrichment in nematode miRNAs associated with the host AGO1, together with
442 the recurrent deployment of highly conserved miRNA families by phylogenetically distant
443 helminth parasites, supports the view that cross-organism small-RNA communication has
444 evolved as a stereotypical virulence strategy. In addition to identifying the nematode miRNAs
445 loaded into plant AGO1, one of the major implications of this work is the apparently
446 convergent use of ancient miRNA families as secreted signals by different parasitic helminths.
447 For instance, the miR-2, miR-71, and miR-100 families are consistently identified in the EVs
448 of diverse parasitic nematodes or in the serum of their host, highlighting their roles as
449 universal effectors of parasitism (Sotillo et al., 2020; Chowdhury et al., 2024). At family
450 level, miRNA repertoires are broadly conserved, but repeated observations of the preferential
451 release and detection of a subset of highly conserved miRNAs, such as members of the miR-2
452 family, in host-associated compartments provides strong evidence against passive leakage,
453 instead suggesting that there is positive selection for cross-species functionality. The situation
454 is different for the highly expressed secreted miR-100 and miR-71. Importantly, convergence
455 does not necessarily imply a conservation of precisely the same target genes. Instead,
456 parasites may repeatedly exploit a limited number of host regulatory hubs, such as that for
457 immunity, through the use of conserved target sites or different species-specific sites with
458 similar physiological outputs. Our data therefore support a model in which cross-kingdom
459 miRNA delivery constitutes a recurrent, evolutionarily reinforced virulence strategy. One
460 limitation of our AGO1-RIP dataset concerns let-7. We excluded AGO1-associated min-let-7
461 as likely contamination because it was also detected in the negative control (non-infected
462 roots incubated with free-living J2s). However, let-7 has been repeatedly reported in
463 extracellular vesicles/secrections and host-associated fluids in multiple parasitic systems,
464 including plant-parasitic nematodes (Sotillo et al., 2020; Ste-Croix et al., 2023), and it co-
465 localizes with the secreted miR-100 in a conserved genomic cluster in M. incognita. Thus,
466 although we opted for a conservative interpretation here, we cannot rule out that let-7 is
467 genuinely secreted and loads into host AGO1 during infection; resolving this will require
468 further analysis.
Together, our data support a working model in which M. incognita implements a highly selective, miRNA-driven cross-kingdom RNAi program during infection. A restricted subset of nematode miRNAs is preferentially sorted for secretion independently of their abundance in the parasite, delivered into root giant feeding cells, and selectively loaded into host AGO1, positioning them within the core plant silencing machinery. A fraction of these AGO1-bound miRNAs then converges on key host regulatory hubs—hormone-mediated immunity, metabolic reprogramming, and cellular organization/cytoskeletal dynamics—thereby coordinating broad transcriptional and physiological outputs that attenuate defense while promoting giant-cell development and sustained feeding-site function across hosts. Consistent with this model, miR-2b emerges as an effector that enhances giant feeding cell formation/maintenance in infected roots. Future work should define the *in vivo* contribution of individual secreted miRNAs, elucidate the mechanisms underlying selective sorting and delivery, and extend analyses to other transferred nucleic acids to capture the full scope of cross-organism molecular exchange during plant–nematode interactions.

483

## 484 METHODS

### 485 Plant Growth Conditions and Inoculation

*Solanum lycopersicum* (cultivar M82) seeds were sown on 0.8% agar plates, and incubated at 22°C in darkness for four days. Seedlings were transplanted into a 1:1 mixture of soil and sand (B5) and grown in a controlled-climate chamber under a 16-hour light/8-hour dark photoperiod, at 22°C and 60% relative humidity.

*Arabidopsis thaliana* (ecotype Col-0) seeds were stratified for 48 hours at 4°C, sown in potting soil and placed in a germination chamber for one week (8-hour light/16-hour dark photoperiod, 22°C, 80% relative humidity). Individual seedlings were transplanted into single soil pots and placed in a growth chamber under an 8-hour light/16-hour dark cycle at 22°C and 60% relative humidity.

One month after germination, each plant was inoculated with second-stage juveniles (J2) of *Meloidogyne incognita* (strain Morelos), 500 such larvae for *Arabidopsis* and 1500 for *S. lycopersicum*. Fourteen days post-infection, galls harvested from infected plants or roots from uninfected plants (as a control), were flash-frozen in liquid nitrogen, and stored at -80°C for further analysis.
500 RNA extraction and sequencing

501 Preparasitic J2 were flash frozen in liquid nitrogen and ground to a powder in a tissue lyser
502 (Retsch; MM301) with 4 mm tungsten balls at a frequency of 30 Hertz for 30 seconds. Total
503 RNA was extracted from these samples with the miRNeasy Mini Kit (Qiagen), according to
504 the manufacturer's instructions, but with three additional washes in RPE buffer.

505 The quality of the small RNAs generated by AGO1-RIP and of the total J2 RNA were
506 checked with a Bioanalyzer High-Sensitivity sRNA kit High-Sensitivity RNA kit
507 respectivelu. Small RNAs and total J2 RNAs were sent to the Beijing Sequencing Institute
508 (Hong Kong, China) for the construction of libraries and sequencing. The full raw sequencing
509 data were submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/).

510 M. incognita MiRNA de novo prediction

511 Two complementary approaches were used for small RNA prediction in the nematode, to
512 increase robustness. First, miRNAs were predicted with miRDeep2 (v2.0.0.8), which was run
513 independently on each sequencing library (Friedländer et al., 2012). Only miRNA candidates
514 detected in at least two independent libraries were retained for downstream analyses. In
515 parallel, miRNA prediction was also performed with ShortStack (v3.8.5) (Axtell, 2013). We
516 used a multi-step strategy to decrease the false-positive rate: (i) candidate loci were defined
517 from the initial ShortStack predictions; (ii) loci were compared across libraries with
518 MultiIntersectBed, retaining only those supported by at least three libraries; and (iii) the
519 retained loci were merged to generate a consolidated locus set. ShortStack was then rerun
520 independently on each library, using this consolidated set, to refine miRNA calls. Finally,
521 miRDeep2- and ShortStack-derived predictions were compared and merged with the aid of a
522 custom Perl script, yielding a final high-confidence set of predicted miRNAs.

523 Co-immunoprecipitation of AGO1-Associated sRNAs

524 Frozen galls were harvested and ground into a fine powder in liquid nitrogen: 5 g from galls
525 of *S. lycopersicum* and 1 g from galls of *Arabidopsis*. We mixed 5 g of root tissue from
526 uninfected tomato plants with *M. incognita* J2 larvae (1000 J2 per gram of fresh uninfected
527 root) as a control. AGO1 immunopurification was performed as described by Dunker et al.,
528 (2021) with a slight modification. Non-specific co-purification was minimized by incubating
529 the extract with Protein A agarose beads (Roche) for 30 min before washing. The beads were
530 removed by centrifugation at 300 x g for 1 min at 4°C. AGO1 immunoprecipitation was
performed by incubating the cleared extract with 1 µg per gram of tissue on a rotating wheel for 4 hours at 4°C. We added Protein A agarose beads at a concentration of 100 µL per 5 mL IP buffer, and the mixture was incubated overnight at 4°C with continuous rotation.

### 534 AGO1-associated small-RNA sequencing analysis

AGO1-immunoprecipitated (AGO1-IP) sRNA libraries were processed with a reproducible bioinformatics pipeline, starting from raw FASTQ files, as follows

(i) Read preprocessing and rRNA depletion: rRNA contaminants were removed by filtering raw reads with SortMeRNA v4.3.6 against the SILVA rRNA database (v138.1) with default parameters (Martinez et al., 2008). The remaining reads were size-selected to retain only sequences of between 18 and 25 nt. We ensured that rRNA depletion was exhaustive by performing a second specific filtering step with Bowtie v1.3.1 (Langmead et al., 2009). Reads were aligned against the SILVA 138.1 database with stringent parameters adapted for short sequences (-l 10 -e 70 -v 0, allowing only perfect matches) (Quast et al., 2013). The non-aligning reads (rRNA-free) were extracted from SAM files with Samtools v1.14 (bam2fq) for downstream analysis (Danecek et al., 2021).

(ii) Genome mapping and quantification: The rRNA-free sRNA reads were mapped onto the reference genomes of *Solanum lycopersicum* (Heinz1706 -v3.00), *Meloidogyne incognita* (v3), and a combined tomato-nematode reference genome with Bowtie v1.3.1. We ensured computational reproducibility by performing all alignments in a Singularity container environment. The following alignment parameters were applied: perfect matches only (-v 0), reporting all valid alignments (-a), and retaining only the best alignments (--best --strata). Aligned sequences were extracted and collapsed into unique tags. The abundance of each unique sequence was quantified across conditions and reference genomes with custom Perl scripts.

(iii) miRNA homology search and annotation: Homology-based annotation was performed on unique sequences with HMMER v3.4 (nhmmer) in DNA mode (Wheeler and Eddy, 2013). Search targets included miRBase, and sets of predicted miRNAs for both *S. lycopersicum* and *M. incognita* (Kozomara et al., 2019). Alignments were optimized for short reads with a score threshold of -T 5 and the F3 filter enabled. Significant hits were retained to generate the final annotation of AGO1-associated sRNAs.
561 (iv) Thresholds of 50 reads per million present in two replicate galls for each sequence were
562 applied and the final list of sRNAs was manually curated with the retention of perfect
563 alignments against the Rfam and Ensembl databases to eliminate all sequences corresponding
564 to tRNA, snoRNA, snRNA, long non coding RNAs (lncRNAs) and all sense mRNAs.

565 Degradome analysis

566 The degradome sequencing of tomato galls (Noureddine et al., 2023) was analysed with
567 CleaveLand 4.0 (Addo-Quaye et al., 2009), using the default parameters and the secreted
568 nematode miRNAs. *In sillico* prediction was performed with psRNATarget (default Schema
569 V2) (Dai et al., 2018), using the list of secreted miRNA and the *Solanum lycopersicum* cDNA
570 library ITAG3.0 as input.

571 Quantification of miRNA Cleavage Activity With the Dual-Luciferase Reporter

572 DLR assays were performed with the DLR Assay System (Promega). For each plant
573 target/nematode amiRNA pair, two leaves from four-week-old *Nicotiana benthamiana* plants
574 were infiltrated with an *Agrobacterium* mixture containing pK2GW7-amiRNA and pGrDL-
575 TS constructs at a 1:5 ratio in infiltration buffer (2 mM MgCl₂, 2 mM MES, 100 µg/mL
576 acetosyringone). The mixture was incubated for 4 hours before infiltration. The leaves were
577 harvested 72 hours after infiltration, ground into a fine powder in liquid nitrogen with 4 mm
578 tungsten beads in n MM400 vibratory crusher (Retsch; MM30) and stored at -80°C. Five mg
579 of ground leaf tissue was resuspended in 250 µL 1X Passive Lysis Buffer (PLB; Promega). A
580 15 µL aliquot of the lysate was diluted in 360 µL buffer for analysis. *Renilla* and firefly
581 luciferase activities were measured with a SAFAS luminometer. Measurements were
582 performed on 96-well plates with 15 µL of leaf extract per well. Firefly activity was measured
583 10 seconds after the injection of 75 µL Luciferase Assay Buffer II (Promega), and then 10
584 seconds after the addition of 75 µL Stop & Glow reagent for *Renilla* luciferase activity
585 (Promega). The mean luminescence ratio over 10 seconds was calculated for each luciferase
586 in the eight biological replicates of each nematode miRNA/tomato target-site pair, with at
587 least two independent experimental replicates. Ratios were normalized against those obtained
588 with the pGrDL plasmid lacking a target site and are presented as log₂ values. Statistical
589 comparisons of F-Luc/R-Luc ratio normalized against pGrDL_SPb and converted into log₂
590 ratios between each pGrDL and the pGrDL_SPb control co-infiltrated with the
591 same amiRNA were performed with unpaired Student's *t*-tests in GraphPad Prism v8.

592 Microsynteny analysis
We used MCScanX (Wang et al., 2012) with default parameters to determine whether the *MIR* regions of genus *Meloidogyne* genus displayed conserved synteny. BLASTp files and GFF3 annotations were provided for all the available *Meloidogyne* species (*M. graminicola*, *M. chitwoodi*, *M. hapla*, *M. enterolobii*, *M. arenaria*, *M. javanica*, *M. incognita* and *M. luci*) and the closest outgroup species, *P. penetrans*. MCScanX was ran with the default parameters. However, the -b interspecies parameter was used to identify collinear blocks between different species, with an e-value of 0.001, and the -s 4 parameter was used to impose a requirement of at least four genes per collinear block. We obtained a set of syntenic blocks alongside a detailed list of syntenic gene pairs, which we used to identify *MIR* regions in the collinearity file and to determine in which *Meloidogyne* species these regions were conserved.

### *S. lycopersicum* hairy-root production and infection assay

Artificial microRNAs (amiRNAs) were expressed transiently in *S. lycopersicum* by *Agrobacterium rhizogenes*-mediated hairy-root transformation. The binary vector pK2GW7-amiRNA was introduced into *Agrobacterium tumefaciens* strain C58C1 harboring the *A. rhizogenes* pRiA4 plasmid and maintained under chloramphenicol, streptomycin and kanamycin selection pressure. Tomato (cv. M82) seeds were surface sterilized by HCl treatment and germinated on Murashige and Skoog (MS) medium for 10 days. Seedlings were then subjected to stem agroinfiltration by injection of the *Agrobacterium* suspension (OD=1) with a syringe. Ten days post-inoculation, stem segments bearing emerging hairy roots were excised and transferred to MS medium supplemented with 3% sucrose, 0.3% phytagel, 0.05% MES, 500 µg/mL ticarcillin, 200 µg/mL cefotaxime, and 30 µg/mL kanamycin for the selection of transformed roots. Hairy roots carrying the pK2GW7-amiRNA construct were verified by PCR and transferred to MS medium without antibiotics. They were allowed to regenerate for one week and individual hairy roots were then inoculated with 250 freshly hatched preparasitic second-stage juveniles (J2s). The J2s were surface-sterilized with HgCl² and treated with streptomycin before inoculation. At 14 days post-inoculation, galls were collected and fixed in 30% formaldehyde in 1x PBS under a vacuum. The tissues were cleared with benzyl alcohol/benzyl benzoate (BABB) and the feeding sites were then measured.
623 Benzyl alcohol/benzyl benzoate clearing and feeding site measurement

624 After one week of regeneration, individual hairy roots were inoculated with 250 freshly

625 hatched sterilized preparasitic J2s. Measurements of giant cell area within 14 dpi galls were

626 performed as previously described (Cabrera *et al.*, 2018; Noureddine *et al.* 2023).

627

628 FUNDING

629 A.D. was supported by 'Sante des Plantes et Environnement' INRAE department and

630 Provence Alpes Cote d'Azur fellowship. This work was supported by a PhD grant from SPE

631 INRAE and PACA Region, by the COST ExRNAPATH program and the UCA JEDI

632 Investments in the Future project (ANR-15-IDEX-566 01) managed by the National Research

633 Agency (ANR).

634

635 AUTHOR CONTRIBUTIONS

636 A.D., S.J., and B.F. conceived the study and designed experiments. Unless otherwise

637 specified, A.D. performed all of the experiments and conducted data analysis. MdR

638 performed the bioinformatic analysis. E.S. performed microsynteny analysis. A.W., Y.N. and

639 A.P.C initiate the AGO1-RIP. K.M. genotyped the hairy roots. P.F. performed the BABB

640 experiments. O.Y. and A.T.M. performed some DLR analyses. J.Z. and L. N. supervised

641 some sRNAs analysis. M.Q. take part to the writing. A.D., S.J. and B.F. wrote the article.

642 ACKNOWLEDGMENTS

643 We would like to thank Etienne Danchin and Cei Abreu for their advices on the RKN genome

644 and microRNA analysis, Sophie Mantelin for her advice on cloning, Julien Pirello and Lydie

645 Tessaroto for their help in setting up the tomato root transformation.

646 FIGURE LEGENDS

647 Figure 1. Nematode small RNAs associated with SLAGO1 in tomato galls at
648 14 dpi.

649 (A) Top 15 most abundant *M. incognita* miRNAs in sRNA-seq libraries from
650 pre-parasitic J2s, shown as mean abundance across three biological replicates in
651 read per million (RPM). (B) *M. incognita* miRNAs detected in association with
652 tomato AGO1 (SIAGO1) in galls at 14 dpi.

653 miRNAs highlighted in red correspond to min-miRNAs detected in association
654 with *S. lycopersicum* AGO1 in galls. (C) SIAGO1-RIP size and 5'-nucleotide
655 profiles from two biological replicates of tomato galls, shown as average (RPM)
656 and number of unique sequences for each sRNA size class.

657 **Figure 2. Experimental validation of nematode miRNA-directed silencing**
658 **of predicted tomato targets.**

659 Nematode miRNAs and their predicted tomato target sites were co-expressed in
660 *Nicotiana benthamiana* leaves to assess silencing activity. Target sites were
661 fused to the firefly luciferase coding sequence in a dual-luciferase reporter
662 (DLR) system, with constitutively expressed Renilla luciferase as an internal
663 control. The log2-transformed Firefly/Renilla (F-luc/R-luc) ratio measured in
664 co-infiltrated leaves was normalized to the negative control. A perfectly
665 complementary target sequence (“Perfect”) served as a positive silencing
666 control. For negative-control assays, a “Spacer” corresponding to the native 5’
667 UTR sequence upstream of firefly luciferase (with little or no complementarity
668 to the tested miRNA) was used. Asterisks indicate statistical significance
669 (unpaired Student’s t-test): *P* < 0.05, *P* < 0.005, *P* < 0.0001.

670 **Figure 3. In planta overexpression of min-miR-2b increases feeding-site size**
671 **in tomato galls.**

672 The effects of min-miR-2b and min-miR-57 on giant feeding cell development
673 were assessed by quantifying the feeding-site area (giant-cell region; outlined in
674 red) in tomato roots transiently expressing miR-2b or miR-57, compared with
675 empty-vector controls. Galls were collected 2 weeks after *in vitro* infection and
676 cleared using the BABB method (Cabrera et al., 2018) to measure the area (µm²)
677 corresponding to giant cells. Differences were evaluated using Mann-Whitney
678 tests ($P$ < 0.025). Box plots show the interquartile range (25th–75th percentiles)
679 with the median indicated by the center line; whiskers represent the minimum
680 and maximum non-outlier values, and points denote outliers. $n$ indicates the
681 number of galls analyzed per line and condition. Scale bar, 100 µm. N,
682 nematode.

683

684 SUPPLEMENTAL INFORMATIONS

685 • Supplemental Figure 1: Characteristics of *M. incognita* mature and precursor miRNA
686 sequences.

687 • Supplemental Figure 2: Experimental test of tomato genes identified as predicted
688 targets of nematode miRNAs.

689 • Supplemental Figure 3: Conservation of the tomato miR-2b target site of SIMT2 in
690 *Solanum lycopersicum* and *Arabidopsis thaliana* methyltransferase-encoding genes.

691 • Supplemental Figure 4: Genome organization and sequence conservation of the miR-2
692 family within the phylum Nematoda

693 • Supplemental Figure 5: MicroRNAs secreted by nematodes, trematodes and cestodes.

694 • Supplemental Figure 6: Western-blot analysis of SIAGO1-IP

695 • Supplemental Table 1: *M. incognita* MIR gene prediction

696 • Supplemental Table 2: Genomic clusters of MIR genes

697 • Supplemental Table 3: miRNA expression in J2 G7 G14

698 • Supplemental Table 4: SIAGO1-RIP sequencing

699 • Supplemental Table 5: Analysis of the differential expression of miRNA in SIAGO1-RIP
700

701 • Supplemental Table 6: Tomato target prediction based on degradome analysis and
702 CleaveLand V4 cat ≤2

703 • Supplemental Table 7: Tomato target prediction with psRNATarget Exp. Score ≤3.5
704 • Supplemental Table 8: Selected *S. lycopersicum* miRNA targets

705 • Supplemental Table 9: AtAGO1-RIP sequencing

706 • Supplemental Table 10: Microsynteny of secreted miRNA in *Meloidogyne* spp.

707 • Supplemental Table 11: List of primers and oligomers used in this study

708 • Supplemental Figure 8: Anti-AGO1 western blot on S1AGO1-IP

709

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870
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure 1. Nematode small RNAs associated with SIAGO1 in tomato galls at 14 dpi.</figcaption></figure>

(A) Top 15 most abundant *M. incognita* miRNAs in sRNA-seq libraries from pre-parasitic J2s, shown as mean abundance across three biological replicates in read per million (RPM). (B) *M. incognita* miRNAs detected in association with tomato AGO1 (SIAGO1) in galls at 14 dpi. miRNAs highlighted in red correspond to min-miRNAs detected in association with *S. lycopersicum* AGO1 in galls. (C) SIAGO1-RIP size and 5'-nucleotide profiles from two biological replicates of tomato galls, shown as average (RPM) and number of unique sequences for each sRNA size class.

<footnote>bioRxiv preprint doi: https://doi.org/10.64898/2026.02.13.705329; this version posted February 16, 2026. The copyright holder has placed a preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, re-use, and adapt this material for any purpose without crediting the original authors.</footnote>
<figure><img src="image_10.png" /><figcaption>Figure 2. Experimental validation of nematode miRNA-directed silencing of predicted tomato targets.</figcaption></figure>

Nematode miRNAs and their predicted tomato target sites were co-expressed in *Nicotiana benthamiana* leaves to assess silencing activity. Target sites were fused to the firefly luciferase coding sequence in a dual-luciferase reporter (DLR) system, with constitutively expressed Renilla luciferase as an internal control. The log2-transformed Firefly/Renilla (F-luc/R-luc) ratio measured in co-infiltrated leaves was normalized to the negative control. A perfectly complementary target sequence (“Perfect”) served as a positive silencing control. For negative-control assays, a “Spacer” corresponding to the native 5' UTR sequence upstream of firefly luciferase (with little or no complementarity to the tested miRNA) was used. Asterisks indicate statistical significance (unpaired Student's *t*-test): *P* < 0.05, **P** < 0.005, ***P** < 0.0001.

This version posted February 16, 2026. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, adapt, or republish this material for any purpose without crediting the original authors.
<table><caption>Table 1: List of SIAGO1-associated *M. incognita* miRNAs in tomato galls at 14dpi.</caption><thead><tr><th>min-miRNA</th><th>Seq AGO1-IP reads</th><th>5' N</th><th>lenght (nt)</th><th>average G14 RPM</th><th>average R+J2 RPM</th></tr></thead><tbody><tr><td>miR-2b</td><td>TATCACAGTTCGATATGGCCTC</td><td>U</td><td>22</td><td>43505</td><td>18</td></tr><tr><td>miR-5359</td><td>TCTGGGATTTTTTATGTTGGTA</td><td>U</td><td>22</td><td>1932</td><td>1</td></tr><tr><td>miR-100</td><td>AACCCGTAGATCCGAACTAGTCT</td><td>A</td><td>23</td><td>882</td><td>0</td></tr><tr><td>miR-92</td><td>TATTGCACTCGTTTCGGCCTGA</td><td>U</td><td>22</td><td>777</td><td>0</td></tr><tr><td>miR-58</td><td>TGAGATCAGTCCAGATTCGTCG</td><td>U</td><td>22</td><td>736</td><td>0</td></tr><tr><td>miR-71</td><td>TGAAAGACATGGGTAGTTGAGACG</td><td>U</td><td>24</td><td>520</td><td>0</td></tr><tr><td>let-7</td><td>TGAGGTAGTAGGTTGTATAGTT</td><td>U</td><td>22</td><td>393</td><td>210</td></tr><tr><td>miR-7904</td><td>TCAAAAATTCCGTTGCGTCGCA</td><td>U</td><td>22</td><td>191</td><td>0</td></tr><tr><td>miR-Miex1</td><td>ACGAATCGTTTGCACATCGGTGTT</td><td>A</td><td>24</td><td>189</td><td>0</td></tr><tr><td>miR-Miex2</td><td>AAGCACCATGTTGAAGATCGAGA</td><td>A</td><td>23</td><td>170</td><td>0</td></tr><tr><td>miR-57</td><td>TACCCTGTAGTCCCGAGCCGTTTG</td><td>U</td><td>24</td><td>159</td><td>0</td></tr></tbody></table>
<table><caption>Table 2: List of targets validated in DLR with gene ID, annotation and prediction method</caption><thead><tr><th>min-miRNA</th><th>Target ID</th><th>Gene description</th><th>Annotation</th><th>Degradome</th><th>psRNA Target</th></tr></thead><tbody><tr><td rowspan="3">miR-2b</td><td>Solyc12g049320</td><td>Nodulation signaling pathway 1-like</td><td>GRAS50</td><td>cat2</td><td>3.5</td></tr><tr><td>Solyc09g091540</td><td>S-adenosyl-L-methionine-dependent methyltransferase</td><td>SIMT2</td><td>cat0</td><td>X</td></tr><tr><td>Solyc06g067890</td><td>Alpha/beta hydrolase-like</td><td>AMAR1</td><td>X</td><td>2.5</td></tr><tr><td rowspan="2">miR-5359</td><td>Solyc11g045110</td><td>Sucrose-phosphate synthase family protein</td><td>SPS4F</td><td>X</td><td>2.5</td></tr><tr><td>Solyc11g006760</td><td>Multiple myeloma tumor-associated protein</td><td>LILR-like</td><td>cat2</td><td>3.5</td></tr><tr><td rowspan="2">miR-7904</td><td>Solyc11g005450</td><td>Transcription elongation factor A protein 3</td><td>Med26c</td><td>cat2</td><td>4.5</td></tr><tr><td>Solyc03g118010</td><td>Nuclease domain-containing protein 1</td><td>TSN1</td><td>X</td><td>2.5</td></tr><tr><td rowspan="2">miR-Miex1</td><td>Solyc07g065890</td><td>Uridine kinase</td><td>UKL4</td><td>cat2</td><td>4.5</td></tr><tr><td>Solyc06g072690</td><td>Mitotic-spindle organizing 1B-like protein</td><td>GCP3</td><td>X</td><td>3.0</td></tr></tbody></table>
<figure><img src="image_11.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.64898/2026.02.13.765926; this version posted February 16, 2026. The copyright holder has placed preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, re-use, and adapt this material for any purpose without crediting the original authors.

<caption>**Figure 3.** *In planta* overexpression of min-miR-2b increases feeding-site size in tomato galls.
The effects of min-miR-2b and min-miR-57 on giant feeding cell development were assessed by quantifying the feeding-site area (giant-cell region; outlined in red) in tomato roots transiently expressing miR-2b or miR-57, compared with empty-vector controls. Galls were collected 2 weeks after *in vitro* infection and cleared using the BABB method (Cabrera et al., 2018) to measure the area (µm²) corresponding to giant cells. Differences were evaluated using Mann-Whitney tests (*P* < 0.025). Box plots show the interquartile range (25th–75th percentiles) with the median indicated by the center line; whiskers represent the minimum and maximum non-outlier values, and points denote outliers. *n* indicates the number of galls analyzed per line and condition. Scale bar, 100 µm. N, nematode.</caption>