Fungal Argonaute proteins act in bidirectional cross-kingdom RNA interference during plant infection

Fungal Argonaute proteins act in bidirectional cross-kingdom RNA interference during plant infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fungal Argonaute proteins act in bidirectional cross-kingdom RNA interference during plant infection Arne Weiberg, An-Po Cheng, Lihong Huang, Lorenz Oberkofler, Nathan R Johnson, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4183067/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Argonaute (AGO) proteins bind to small RNAs to induce RNA interference (RNAi), a conserved gene regulatory mechanism in animal, plant, and fungal kingdoms. Small RNAs of the fungal plant pathogen Botrytis cinerea were previously shown to translocate into plant cells and bound to the host AGO, which induced cross-kingdom RNAi to promote infection. However, the role of pathogen AGOs during host infection stayed elusive. In this study, we revealed that members of fungal plant pathogen Botrytis cinerea BcAGO family contribute to plant infection and act in bidirectional cross-kingdom RNAi, from fungus to plant and vice versa . Providing these new mechanistic insights of pathogen AGOs promise to improve RNAi-based crop protection strategies. Biological sciences/Molecular biology/RNAi Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Argonautes (AGOs) belong to a conserved protein family and are key compounds of the RNA interference (RNAi) pathway, directing (post-) transcriptional gene silencing in animals, plants, and fungi. AGOs comprise a modular structure of PAZ, MID, and PIWI protein domains that are functional and crucial in RNA binding and gene silencing 1 . In plants, different AGO proteins bind to distinct classes of regulatory small RNAs, small-interfering (si)RNAs and micro(mi)RNAs, to form a ribonucleoprotein complex, known as the RNA-induced silencing complex (RISC). Previous studies revealed key biological functions of AGO proteins for the post-transcriptional regulation of RNAs and the remodeling of heterochromatin at the DNA level to fine-tune gene expression, as well as for the silencing of transposons and viruses 2 . In fungi, AGOs have been well characterized in non-pathogenic contexts. The Neurospora crassa AGO named QDE2 is required for RNAi in the quelling process 3 , 4 . Another N. crassa AGO, named SMS-2, mediates meiotic silencing of unpaired DNA (MSUD) during sexual reproduction 5 . In the basidiomycete Cryptococcus neoformans , sex-induced silencing (SIS) is another RNAi mechanism that depends on the fungal AGO1 6 . RNAi-related heterochromatic silencing of centromere regions is dependent on AGO1 in the fission yeast Schizosaccharomyces pombe 7 . Fungal AGO proteins contribute to transposon and transgene silencing, defense against mycovirus, endogenous gene regulation, and DNA repair 8 , 9 , 10 , 11 . Studies with ago loss-of-function mutants in diverse fungal species revealed AGO involvement in regulating metabolic processes as well as affecting fungal growth, differentiation, development, and pathogenicity 12 , 13 , 14 . Botrytis cinerea is a destructive fungal plant pathogen that infects more than 1,400 different plant species and causes the grey mold disease in many economically important crops 15 , 16 . B. cinerea secretes small RNAs (BcsRNAs) into plant cells that bind to the plant`s own AGO1 to manipulate host immunity genes 17 , 18 ; a virulence mechanism called cross-kingdom RNAi 19 . The biogenesis of cross-kingdom BcsRNAs requires the RNA-dependent RNA polymerase (RDR)1 and two Dicer-like (DCL) proteins 20 , 21 , while B. cinerea bcdcl1bcdcl2 and bcrdr1 knockout mutants exhibited reduced infectivity 18 , 20 , 21 . Cross-kingdom and trans-species RNAi have been reported in distinct pathogenic and mutualistic host-interacting organisms, including fungi, oomycetes, bacteria, parasitic plants, and nematodes 18 , 22 , 23 , 24 , 25 , 26 , 27 , 28 . Moreover, cross-kingdom RNAi is bidirectional, because plants send small RNAs into interacting species to defend themselves against fungal pathogens 20 , 29 , 30 . In this study, we identified and characterized the B. cinerea AGO protein family during infection of the host plant Solanum lycopersicum (tomato). Intriguingly, bcago1 knockout mutants failed to induce cross-kingdom RNAi similar to bcdcl1dcl2 and bcrdr1 mutants 21 ; however, bcago1 was not impaired in virulence. Our data show that BcAGO1 mediates bidirectional cross-kingdom RNAi, from fungus to plants and vice versa . In contrast, BcAGO2, which is also involved in cross-kingdom RNAi, is a crucial pathogenicity factor. Lastly, we present evidence that BcAGO1, BcAGO2, and BcAGO3 control endogenous mRNA expression in B. cinerea . Our study reveals the diversified regulatory functions of different BcAGOs and contributes to our understanding of the molecular mechanisms of plant infection by this economically important pathogen. Results Argonautes in the fungal plant pathogen B. cinerea In this study, we aimed to investigate the role(s) of B. cinerea BcAGOs during tomato infection. We performed a BLASTp search using the full-length protein sequence of the well-characterized N. crassa QDE2 as a query in the genome sequence of the B. cinerea strain B05.10 31 to identify BcAGOs. Four BcAGO proteins that we termed BcAGO1, BcAGO2, BcAGO3, and BcAGO4 were predicted to comprise conserved PAZ and PIWI domains (Figure S1 A). The MID domain and an AGO-conserved N-terminal domains were also found in the BcAGO1 and BcAGO2 using InterPro 32 . The PIWI domain of all four putative BcAGOs contained a conserved aspartic acid (D)/ glutamic acid (E)/D/ histidine (H) catalytic tetrad (Figure S2 ), suggesting that BcAGOs could function as slicer proteins 1 , 33 . We classified the four BcAGOs regarding functional homology by performing phylogenetic analysis including AGO amino acid sequences of different ascomycete species and the well-characterized Arabidopsis thaliana AtAGO1 and Homo sapiens HsAGO2 (Figure S1 B, Table S1 ). The phylogenetic tree positioned BcAGO1 into the fungal quelling clade and the BcAGO2 into the fungal MSUD clade. BcAGO3 and BcAGO4 laid outside these two clades and were of unknown homologous function. The coding gene sequence of BcAGO4 for the B. cinerea strain B05.10 (ASM83294v1) included a predicted premature stop codon in the third exon (Figure S3 A), which suggested that BcAGO4 is a pseudogene. The stop codon likely resulted in the automated annotation of the N- and the C-terminal part of BcAGO4 into two separate genes, Bcin15g05050 and Bc15g05060 , as deposited in the Ensembl database. This point mutation was not found in genome sequences of two other released B. cinerea genome sequences of the strains T4 (GCA_000227075) and BcDW1 (GCA_000349525), respectively (Figure S3 B). To exclude the possibility that this mutation was an error introduced during whole genome sequencing, we cloned BcAGO4 from the B05.10 strain and confirmed this mutation being present by Sanger sequencing (Figure S3 C). This shows that there is within-species genetic variation at the BcAgo4 locus among B. cinerea strains. In the following experiments of this study, we used the strain B05.10. Accumulation of fungal cross-kingdom small RNAs is controlled by BcAGO1 We first explored the possibility that BcAGOs might control BcsRNA accumulation. For this, we generated bcago ko mutants and isolated two single knockout (ko) mutant strains for each BcAGO, referred to as bcago1 , bcago2 , bcago3 , bcago4 (Figure S4 A). We performed stem-loop reverse transcription (RT) PCR of BcsRNAs, collecting samples of B. cinerea grown in axenic culture and from infected tomato leaves (Sl-infected). We chose the BcsRNA3.1, BcsRNA3.2, BcsRNA20, because these were previously found to induce cross-kingdom RNAi of tomato genes during B. cinerea infection 18 , 21 , 34 . We determined that bcago1 ko mutants and a bcago1ago2 double-ko (Figure S4 A) lost the accumulation of BcsRNA3.1, BcsRNA3.2, and BcsRNA20 (Fig. 1 A, Figure S5 ). BcsRNA accumulation was reconstituted in a 3xHA-tagged BcAGO1 complementation strain (Figure S4 B, Figure S6 ). We next performed comparative small RNA deep sequencing analysis to profile global changes of BcsRNA accumulation. Two independent strains of each bcago ko mutant and B. cinerea wild type (WT) were grown in axenic culture for small RNA extraction and Illumina library preparation. Upon raw read processing, we mapped reads to the B. cinerea reference genome allowing a maximum of one mismatch. These BcsRNAs mapped either one or multiple times, when we chose a single “best” map-location with multiple-mappers 35 (Figure S7 A). BcsRNA reads mapped to various annotated genomic regions (Figure S7 B) and showed an overall size enrichment for 21–22 nt reads (Fig. 1 B). An exception was bcago1 , which lost most of the 21–22 nt BcsRNAs that were mainly derived from retrotransposons (Figure S7 B). To be able to measure quantitative differences in BcsRNA accumulation, we annotated for the first time BcsRNA producing genomic loci in B. cinerea , using one of the B. cinerea WT small RNA-seq datasets. We defined BcsRNA loci as regions with coverage above the chromosomal average, defined by a Poisson probability P < 10 − 4 . Nearby regions (< 150 bases apart), given a 10-nucleotide buffer on both sides (Fig. 1 C, Figure S7 C). With these criteria, we identified in total 4,397 BcsRNA loci (Table S2 ), covering 96.3% of the mapped BcsRNAs (Figure S7 D). Annotated loci include many which were not size-specific and are likely derived from but not limited to mRNA degradation (Figure S7 E). Most of the 21–22 nt size-specific BcsRNA loci were found in non-annotated genomic regions, nearby genes, and within gene bodies of exonic and intronic regions. More than 50% of these 21–22 nt size-specific BcsRNA loci overlapped with retrotransposons (Fig. 1 D). As non-size-specific BcsRNAs derived from mRNAs were likely degradation products and not BcAGO-associated, these served as controls for normalization of size-specific BcsRNAs (Figure S8A-B). All differentially expressed BcsRNA loci identified by pairwise comparison between bcago ko mutants and B. cinerea WT are listed in Table S3 . Normalized read counts of BcsRNA loci confirmed that bcago1 ko mutants showed the strongest change in BcsRNA expression, with mostly decreased 21–22 nt size-specific BcsRNA loci expression (Fig. 1 E, Figure S8C). Therefore, bcago1 exhibited reduced cross-kingdom small RNA accumulation, similar to bcrdr1 and bcdcl1dcl2 double-ko mutants, as reported in previous studies 20 , 21 . BcAGO1 is required for fungal-induced cross-kingdom RNAi Since bcago1 ko mutants lost accumulation of 21–22 nt BcsRNAs, we anticipated that bcago1 ko mutant might be compromised in cross-kingdom RNAi during plant infection. We recently developed a GFP “switch on” cross-kingdom RNAi reporter system carrying the BcsRNA3.1 and BcsRNA3.2 target sites of A. thaliana genes. When infecting transgenic A. thaliana reporter plants with B. cinerea , GFP expression is turned on within 24–48 hpi 21 . We used this A. thaliana reporter line to further inspect cross-kingdom RNAi with the bcago1 and bcago2 ko mutants. We infected seedlings with B. cinerea WT, bcago1 , and bcago2 and recorded GFP signal expression in infected leaves in a time course by fluorescence microscopy (Fig. 2 A). This analysis indicated some background fluorescence intensity (I 0 ) in the reporter plants. Therefore, we calculated relative increase of GFP fluorescence intensity (I t -I 0 ) at different time points of infection, following a previously reported analysis 21 . Inoculation with bcago1 ko strain did not induce GFP signal at any measured time point, while bcago2 caused an increase in GFP signal intensity, but to a lesser extent than B. cinerea WT (Fig. 2 B). These observations were corroborated in an immunoblot analysis of GFP protein levels of infected leaves (Fig. 2 C, Figure S9). We measured B. cinerea genomic DNA levels in the A. thaliana reporter plants at the same time point as the GFP measurements and observed no difference between WT, bcago1 , or bcago2 inoculations (Fig. 2 D). This suggested that the observed lower GFP activation was not due to a diminished B. cinerea colonization. Based on the observation, we concluded that BcAGO1 was required for and BcAGO2 also contributed to cross-kingdom RNAi. We next measured mRNA levels of the known tomato BcsRNA3.1, BcsRNA3.2, and BcsRNA20 target genes, namely SlVPS, SlMPKKK4 , and SlBhlh63 21, 34 . SlMPKKK4 and SlBhlh63 were no longer suppressed upon infection with bcago1 ko strains (Fig. 2 E), agreeing with the loss of BcsRNA3.2 and BcsRNA20 accumulation and previous findings that the bcrdr1 also lost BcsRNA accumulation and showed no longer suppression of these target genes upon infection 21 . Target gene suppression was reconstituted upon infection with a 3xHA-BcAGO1 complementation strain. SlMPKKK4 and SlBhlh63 showed similar expression levels upon infection with bcago2 and B. cinerea WT. SlVPS expression was suppressed upon infection with bcago1 at similar levels as B. cinerea WT, but SlVPS was not suppressed upon infection with bcago2 , indicating that this gene was suppressed by a BcAGO2-dependent BcsRNA. All three tomato target genes were no longer suppressed upon infection with a bcago1ago2 double-ko. These results confirmed that both, BcAGO1 and BcAGO2, might be involved in cross-kingdom RNAi, but act on different plant target genes. The tomato immunity marker gene Sl-Proteinase Inhibitor (PI)-II was used as a non-target gene control. Sl-PI-II was more strongly induced when infecting with bcago2 ko mutants compared to B. cinerea WT or bcago1 ko (Fig. 2 F). MPKKK4 is part of a conserved plant immune signaling cassette that involves the downstream targets MPK3/MPK6 and WRKY33 36 . When infecting tomato with bcago1 ko mutants, SlMPK3 and SlWRKY33 were higher expressed compared to infection with B. cinerea WT or bcago2 (Figure S10A). Accordingly, infected tomato revealed higher ROS accumulation upon bcago1 infection compared to B. cinerea WT or bcago2 ko (Figure S10B). Thus, bcago1 and bcago2 mutants were hampered in host immune suppression, but of different immunity pathways. B. cinerea BcAGO2 is a fungal pathogenicity factor To gain further information on the functional role of BcAGOs during tomato infection, we measured BcAGO mRNA levels in axenic culture and in Sl-infected sample conditions by quantitative reverse transcription PCR (qRT-PCR) (Fig. 3 A). BcAGO1 was more highly expressed under both conditions compared to BcAGO2 , BcAGO3 , and BcAGO4 . The BcAGO2 and BcAGO3 displayed up-regulation at 1 day post inoculation (dpi). BcAGO4 showed low expression in both conditions. We next compared disease severity induced by bcago ko mutants using a tomato leaf infection assay. Using conidiospore drop (Fig. 3 B) or agar plug inoculation methods (Figure S11), we observed that bcago2 ko mutants induced smaller lesion areas compared to WT. The bcago1ago2 double-ko showed reduced virulence, similar to bcago2 single ko. Conversely, reduced pathogenicity of bcago2 was reverted in 3xHA- BcAGO2 strains (Fig. 3 B, Figure S11A). Accordingly, we measured significantly less B. cinerea genomic DNA, a proxy for pathogen biomass, in leaf tissue infected with the bcago2 (Fig. 3 C). When growing bcago ko mutants on agar plates, we observed normal fungal growth and development in all strains (Fig. 3 B, Figure S12). Based on these results, we concluded that BcAGO2 is a pathogenicity factor in B. cinerea and hampered cross-kingdom RNAi could explain the reduced pathogenicity. Surprisingly, bcago1 ko mutants did not display any noticeable change in tomato infection, although bcago1 was no longer inducing cross-kingdom RNAi and was hampered in suppressing tomato immunity. Thus, we speculated that BcAGO1 must have a second function during plant infection, and this could be to bind plant-derived small RNAs that could trigger silencing of fungal genes. BcAGO1 facilitates tomato-induced cross-kingdom RNAi Tomato small RNAs might be delivered during infection into B. cinerea cells and hijacked BcAGO1 to suppress fungal pathogenicity genes as a defense response. A previous report showed that A. thaliana delivers small RNAs into B. cinerea to silence pathogenicity-related genes 29 , but a role of BcAGOs was not explored. To identify tomato small RNAs that bind to BcAGOs during infection, we performed BcAGO co-immunopurification (BcAGO IP) coupled to small RNA deep sequencing. For this experiment, we used transgenic B. cinerea strains expressing 3xHA-tagged BcAGO1 or 3xHA-BcAGO2 in the bcago1 or bcago2 ko mutant background, respectively. As observed previously in this study, the transgenic 3xHA-BcAGO1 strains could revert the loss of BcsRNA accumulation in the bcago1 and 3xHA-BcAGO2 reverted the reduced disease phenotype of the bcago2 ko strains, confirming that both 3xHA-BcAGO constructs were functional. Upon immunoblot confirmation of successful BcAGO IP (Figure S13), we isolated small RNAs for cloning and Illumina-based sequencing. We sequenced two biological replicates from axenic culture and three biological replicates for Sl-infected samples (Figure S14A). Small RNA sequencing analysis revealed a shift of size enrichment from 24 nt reads in axenic culture to 21–22 nt reads in Sl-infected samples for both BcAGO1 and BcAGO2 IPs (Fig. 4 A). When mapping reads to the B. cinerea or the tomato reference genomes, we identified sequences that exclusively mapped to one or the other species with at least one mismatch to the opposite reference genome, accordingly. For BcsRNAs, 21–22 nt long retrotransposon-derived reads were enriched in Sl-infected samples (Fig. 4 B). The cross-kingdom BcsRNA3.1, BcsRNA3.2, BcsRNA5, and BcsRNA20, which are all derived from retrotransposons 18 , indicated higher read numbers in Sl-infected samples compared to axenic culture (Fig. 4 C). This result was consistent with a previous finding that retrotransposon-derived 21–22 nt BcsRNAs are induced upon plant infection 34 . Likewise, when mapping reads to our defined BcsRNA loci, we observed overlapping BcsRNA accumulation between BcAGO1 and BcAGO2 (Figure S14B-D) and a size preference of 21–22 nt long reads in size-specific loci (Fig. 4 D). Among BcAGO-bound tomato small RNAs (SlsRNAs), 21 nt long reads were enriched in the BcAGO1 and BcAGO2 IPs (Fig. 5 A). Remarkably, SlsRNAs reached up to 50% of mapped read counts (Fig. 5 B), indicating massive invasion of tomato small RNAs into BcAGOs during infection. The SlsRNA reads mapped to different annotated genetic loci, with the vast majority of them being associated with DNA repeats, mRNAs, tRNAs, and rRNAs (Figure S15). Similar to the BcAGO-bound BcsRNA fraction, the SlsRNAs largely overlapped in binding to BcAGO1 and BcAGO2 during infection (Figure S14D). Among most abundant SlsRNAs, higher read numbers were counted in BcAGO1 IP samples compared to BcAGO2 IP (Figure S16A). We next investigated the possibility that SlsRNA candidates silence B. cinerea genes during infection. We considered 21–22 nt SlsRNA reads with an average of > 100 RPM in the BcAGO1 or BcAGO2 IP small RNA-seq datasets. We filtered out all SlsRNA reads mapping to ribosomal RNA (rRNA), small nuclear or nucleolar RNA (snRNA/snoRNA), or protein coding transcripts (in sense orientation), as these did not likely represent regulatory RNAs but RNA degradation products. In total, we predicted 74 B. cinerea target genes of 21 SlsRNA candidates using the TAPIR tool with stringent parameter setting (Table S4 ). These SlsRNA candidates were detected in all BcAGO IP datasets (Figure S16A). We chose a subset of 10 SlsRNAs to confirm that these invaded into B. cinerea during tomato infection. For this, we re-isolated B. cinerea from Sl-infected leaf tissue to subculture on agar plates (Fig. 5 C). B. cinerea subculture samples were taken from the colony edge after 20 h or 7 d for SlsRNA analysis. The SlmiRNA159, SlmiR162, SlsRNA4, SlsRNA11, and SlsRNA12 were detected in independent B. cinerea re-isolation samples by stem-loop RT-PCR (Fig. 5 C, Figure S16B). These SlsRNAs were not detected in B. cinerea mycelium that was not re-isolated from infected tomato but were present in tomato samples. SlsRNAs were also detected in re-isolated bcago1 , bcago2 and bcago1ago2 ko mutant strains, as well as in a bcrdr1 ko mutant strain that was recently characterized to be required for BcsRNA accumulation and cross-kingdom RNAi 21 (Figure S16C). In this RT-PCR assay, the SlmiR399 served as a negative control SlsRNA, because it was not detected in the BcAGO IP small RNA datasets of this work. To further analyze if SlsRNAs could suppress B. cinerea target genes through BcAGOs during infection, we measured mRNA expression of predicted target genes in Sl-infected samples, comparing B. cinerea WT with bcago1 , bcago2 , and bcago1ago2 ko strains. Among 14 tested B. cinerea candidate genes, Bcin12g05240 , Bcin05g04730 , Bcin07g06910 , and Bcin04g05820 displayed higher expression in bcago1 and bcago1ago2 ko mutants (Fig. 5 D, Figure S17A). Failed mRNA suppression was not due to bcago1 loss-of-function per se , because elevated mRNA expression was not evident when comparing B. cinerea WT and bcago1 ko strains grown in axenic culture (Figure S17B). Moreover, Bcin05g04730 was transcriptionally upregulated in Sl-infected samples upon B. cinerea WT infection, indicating that this gene might be relevant for fungal infection (Figure S17C). The Bcin05g04730 encodes for a putative Serine/Threonine Protein kinase. Hence, we chose Bcin05g04730 for targeted gene ko to assess its potential role in tomato infection. Two independent ko strains (Figure S18) displayed reduced lesion area induction when infecting tomato and strains grew more slowly on agar plates (Fig. 5 E). Thus, we uncovered a novel function of Bcin05g04730 in fungal pathogenicity. B. cinerea BcAGOs regulate fungal mRNAs related to plant infection Apart from cross-kingdom RNAi, BcAGOs could regulate expression of endogenous fungal genes related to pathogenicity. To identify such genes, we conducted a mRNA-seq experiment comparing B. cinerea WT and bcago ko mutant strains grown in axenic culture. PCA of all mapped read counts verified overall sufficient reproducibility of datasets (Figure S19A). Pairwise comparison of B. cinerea WT versus bcago ko mutants revealed differentially expressed genes (DEGs) using cut-off criteria of one-half fold-change expression (log fold change > = 0.58) and adjusted p-value of 0.1 (Table S5 ). In the bcago1 ko mutant, many up-or down-regulated genes were annotated to have extracellular enzymatic activities and comprised known or predicted virulence factors (Figure S20A). In the bcago2 background, we confirmed that Bcin02g00620 , Bcin02g01230 , and Bcin09g00460 were also differentially expressed during tomato infection (Figure S20B) indicating putative functions in pathogenicity. The largest number of DEGs (337 up- and 400 down) were found in the bcago3 ko mutant (Figure S19B) which included many genes belonging to the phytotoxins Botrydial and Botcinic acid biosynthetic gene clusters (Figure S20C). Collectively, in this study, we discovered complex RNA regulation executed by different BcAGOs during host infection in at least three different functions: i) BcAGO1 is required for fungal induced cross-kingdom RNAi by controlling BcsRNA accumulation, ii) BcAGO1 binds SlsRNAs that suppress fungal pathogenicity genes, iii) BcAGO1, BcAGO2, and BcAGO3 regulate endogenous mRNA expression including fungal pathogenicity genes (Figure S21). Discussion Cross-kingdom RNAi is an emerging field in host-pathogen interaction research and bidirectional cross-kingdom RNAi has been described in fungal-plant interaction. In one direction, B. cinerea secretes BcsRNAs into its host plants tomato and A. thaliana that bind to the plants´ own AGO1 to silence host immunity genes 17 , 18 , 34 . In the counter-direction, A. thaliana secretes small RNAs that can trigger gene suppression in B. cinerea 29 . The role of pathogen AGOs in cross-kingdom RNAi had not been explored. We herewith provide new mechanistic insights of the diversified regulatory functions of different BcAGO family members in bidirectional cross-kingdom RNAi and in plant infection. Profiling the small RNA transcriptome revealed that BcAGO1 is required for the accumulation of cross-kingdom BcsRNAs. Reduction but not complete loss of small RNA has been reported in ago loss-of-function mutants before 37 , 38 . It is argued that small RNAs bound to AGO could experience transient protection against rapid nuclease degradation. Alternatively, BcAGO1 might regulate BcsRNA biogenesis. For instance, the fungal Magnaporthe oryzae MoAGO2 interferes with RNAi triggered by hairpin and retrotransposon-derived small RNAs through the MoAGO1 and MoAGO3 39 . A similar BcAGO1-mediated RNAi feedback loop might also exist in B. cinerea . When using a GFP switch-on reporter assay in planta , which allowed us to measure fungal-induced cross-kingdom RNAi in plants over a time course of infection, the bcago1 ko mutant failed in reporter activation. This is in agreement with a previous finding that B. cinerea bcrdr1 and bcdcl1bcdcl2 ko strains, which were impaired in cross-kingdom BcsRNA production 18 , 20 also failed to induce cross-kingdom RNAi in the same reporter plant 21 . Using this assay, we also observed that the bcago2 ko mutant was compromised in cross-kingdom RNAi. However, BcsRNA accumulation was largely unaltered in the bcago2 , implying another role of BcAGO2 in cross-kingdom RNAi, compared to BcAGO1, BcDCLs, and BcRDR1. For instance, A. thaliana AGO1 participates in packaging small RNA into extracellular vesicles (EVs) for secretion 40 . EVs are also produced by B. cinerea that contain BcsRNAs and are taken up into A. thaliana cells via clathrin-mediated endocytosis 41 . Therefore, BcAGO2 might play a role in BcsRNA sorting for secretion, which could explain compromised cross-kingdom RNAi when infecting reporter plants with bcago2 ko. Using bcago ko mutants in a tomato leaf infection assay, we observed reduced pathogenicity in the bcago2 mutant. Likewise, single ago gene ko in the apple canker fungus Valsa mali reduced virulence 42 . An ago ko in the wheat-infecting fungus Zymoseptoria tritici led to stop the production of asexual propagules in planta 13 . A possible reason for the reduced pathogenicity in bcago2 could be the limited capability to induce cross-kingdom RNAi, but it would be necessary to further explore this causality in detail. Cross-kingdom RNAi was reported in both directions during B. cinerea -plant interaction 20 . We here found evidence that BcAGO1 was exploited for tomato-induced cross-kingdom RNAi. By profiling the BcAGO-bound small RNA repertoire during tomato infection, we identified at least 21 tomato SlsRNAs that were predicted to target 74 B. cinerea mRNAs. We demonstrated that four B. cinerea genes were suppressed during infection in a BcAGO1-dependent manner and proofed that one target gene, a serine/threonine protein kinase, was part of B. cinerea pathogenicity. Therefore, studying pathogen AGO-associated host small RNAs during infection, not only deciphered tomato small RNAs that were capable to induce cross-kingdom RNAi, but also promise to reveal novel B. cinerea pathogenicity factors. Interestingly, protein kinases were also previously identified as cross-kingdom RNAi targets of B. cinerea 18 and the oomycete Hyaloperonospora arabidopsidis small RNAs 22 in A. thaliana and tomato host plant species, revealing that manipulating this class of enzymes has evolved in diverse biotic interactions. Differential mRNA transcriptome analysis suggests that BcAGOs regulate endogenous fungal genes related to pathogenicity. As an example, up-regulation of a predicted MFS transporter ( Bcin02g00620 ) during tomato infection is dependent on BcAGO2. It is known that MFS transporters provide resistance to various fungitoxic compounds and regulate B. cinerea tolerance to glucosinolate-breakdown products, which is required for pathogenicity 43 . BcAGO3 regulates expression of several phyotoxin biosynthesis genes belonging to the Botrydial (BOT) and Botcinic acid (BOA) gene clusters 44 , 45 . Previous findings also showed that AGO and other RNAi components in the fungal pathogen F. graminearum are required for mycotoxin deoxynivalenol production and full virulence 12 , suggesting that regulatory function of fungal AGOs in toxin production is conserved. In summary, we uncovered diversified functions of BcAGOs during plant infection, including regulation of fungal endogenous genes as well as fungal- and plant-induced cross-kingdom RNAi. This led to new mechanistic insights into the complex regulatory roles of pathogen AGOs during plant infection. Translating such knowledge into innovative siRNA tools promise to improve RNAi-based crop protection strategies in future. Materials and methods Fungal and plant materials B. cinerea Pers. Fr. ( Botryotinia fuckeliana [de Bary] Whetzel) strain B05.10 46 was used in the study and was cultured in complete HA media 47 , if not otherwise notified. B. cinerea strains were cultivated at 20°C under constant irradiation for conidiation or under constant dark condition. Sporulated mycelia of B. cinerea were eluted with distilled water and filtered by Miracloth (Merck Millipore) to collect conidiospore suspension. Conidia were stored in 25% glycerol and kept at -80°C for a long-term storage. Mycelial plugs (Ø=0.4 cm) were collected in distilled water at 4°C for a temporary storage. Mycelia collected from Sl-infected leaves were stored for inoculation assay to activate fungal virulent genes. Solanum lycopersicum (cultivar Heintz 1706) was grown in a climate chamber under controlled condition (16 h light/8 h dark, 24°C, 60% relative humidity). Arabidopsis thaliana ecotype Columbia (Col)-0 was grown under short-day conditions (8 h light/16 h dark, 22°C, 60% relative humidity). Fungal transformation Botrytis transformation was performed as previously described 48 with minor modifications. Botrytis protoplasts were mixed with SH agar (0.6 M sucrose, 5 mM Tris-HCl (pH 6.5), 1 mM (NH 4 )H 2 PO 4 , 8 g/L agar) without any antibiotics and incubated in darkness for 24 h after transformation. Another layer of SH agar containing hygromycin B (50 µg/ml) or nourseothricin (50 µg/ml) was added to the top after pre-incubation. The plates were kept in darkness for 3–5 days until single colony isolation. Fungal growth assay For observation of the growth rate of Botrytis wild-type and transgenic strains, a droplet of a suspension (20 µl, 5x10 4 spores/ml) with spores in distilled water was pipetted on HA agar media. Petri dishes were incubated for 5 days at room temperature. Mycelial growth was determined by measuring the radial growth of colonies. Conidia were collected from HA media culturing for two weeks under constant light to observe the conidial shape of Botrytis wild-type and transgenic strains. Conidium width and length were measured from over 200 spores for each transformant. Conidia were eluted in distilled water by stirring sporulated mycelium on media plates. The number of conidia produced was determined by counting the spores microscopically with the hemocytometer (Neubauer improved, Marienfeld). Each genotype had six biological replicates. Infection assays For infection assay with spores, conidia were eluted from sporulated B. cinerea with 1% malt extract 47 suspension buffer. A droplet of 20 µl (5x10 4 spores/ml) conidial suspension were inoculated on detached leaves of six weeks old tomato plants. Inoculation assay with mycelial plugs (Ø=0.4 cm) was performed according to the previous method 49 . Mycelia on agar plates of B. cinerea were inoculated on detached leaves of four weeks old tomato plants. The lesion area was measured using Fiji software (ImageJ version 2.1.0/1.53c). For quantifying mycelial growth of B. cinerea , leaf discs three infected leaves were collected for genomic DNA extraction and qRT-PCR by using SYBR Green (Thermo Scientific) with qPCR cycler (CFX96, Bio-Rad). Reactive oxygen species (ROS) measurements ROS detection was performed by luminol-based assay as previous study 50 . Infected tomato leaf discs were collected at 12 hpi and incubated in 96-well plates with 200 µl of sterile water for 12 h in the dark. The water was replaced with 100 µl of working solution (200 µM luminol L-012, 10 µg/ml horseradish peroxidase). 100 nM Flg22 was used as positive control. Luminescence was detected using Photek camera over an hour. Plots are averages of six or eight leaf disks from independent leaves for two independent replicates. DNA and RNA extraction Genomic DNA of pure mycelium or sporulated mycelium was isolated from at least 30 transformants for each construct using CTAB according to the previous method 51 prior to chloroform/isoamyl alcohol extraction and isopropanol precipitation 52 with minor modifications 53 . Mycelia growing on HA media dishes were harvested after cultivation for five to seven days in constant light or overlayed with cellophane for three days under constant dark condition. Six pairs of primers were used for genotyping transgenic transformants for each genotype of B. cinerea . GoTaq G2 DNA Polymerase (Promega) was used for genotyping with cycler. Primer oligos used for genotyping were listed in Table S6 . RT-PCR Six-week-old tomatoes were treated with conidial suspension (2x10 5 spores/ml) of Botrytis wild-type or ko mutant strains using a versatile sprayer (Roth Labware ®). Tomato or A. thaliana leaf discs were collected after 24, 48, 72 hours of treatment for AGO gene expression analysis. Four leaf discs (Ø=0.4 cm) were collected as one biological replicate. Botrytis wild-type and genetically modified strains were cultivated on HA plates overlayed with cellophane for three days in the dark or grown on HA media for seven days under constant light for sporulation. Mycelia from the same plate were collected as a biological replicate. DNA-free total RNAs were used for first-strand cDNA synthesis with oligo (dT) and SuperScript III reverse transcriptase (Invitrogen, Thermo Fischer Scientific) according to the manufacturer’s instructions. RT reactions were diluted 10 folds with ddH 2 O prior to performing qRT-PCR using SYBR Green (Invitrogen, Thermo Fischer Scientific). B. cinerea Tubulin (BCIN_01g08040) was used as reference genes to normalize mRNA. Relative transcripts were calculated by 2 −∆∆Ct based on the previous method 54 using qPCR cycler (Quantstudio5, Thermo Fisher Scientific). Primer sequences were listed in Table S6 . DNA-free total RNAs (1µg) were used for a first-strand cDNA synthesis reaction with specific stem-loop RT primer and reverse transcription was carried out as described previously 55 with minor modifications. The resultant cDNA was directly used for amplification using GoTaq DNA Polymerase (Promega) on a thermo cycler initiated with 95°C for 2 min, then 32 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 20–180 sec (1 kb/min), followed by 5 min extension at 72°C. PCR products were visualized with 10% non-denaturing PAGE gel. Oligonucleotides were provided in Table S6 . BcAGO IP B. cinerea strains expressing 3xHA-BcAGO were grown in liquid HA media overnight. 5 g of fresh mycelia were homogenized by mortar and pestle and suspended in 20 ml extraction buffer according to the previous method (20 mM Tris-HCl pH7.5, 300 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 5 mM DTT, 1 tablet cOmplete® protease inhibitor cocktail (Roche)/50 ml, 5 µl RNase inhibitor (40 U)/50 ml) 56 . The lysate was incubated on a vertical wheel and centrifuged for 40 min at 4°C. Mycelia debris was excluded by spinning down and filtered through Miracloth. EZview™ Red Anti-HA Affinity Gel (Merck) was used for IP. Samples were incubated at 4°C for 1 h. Beads were pelleted down in a pre-cooled centrifuge at 200 × g and washed five times with Wash buffer (20 mM Tris-HCl pH7.5, 300 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X-100, 5 mM DTT, 1 tablet cOmplete® protease inhibitor cocktail/50 ml, 5 µl RNase inhibitor (40 U)/50 ml). Proteins were separated with 8% SDS-polyacrylamide gel at 80 volts for 30 min and 140 volts for 2 h and transferred to PVDF membrane (Immobilon-FL) overnight at 4°C. Transferred membranes were blocked with 10 ml of 5% (v/v) skim fat milk in 1× PBS at 4°C for 1 h on a rolling shaker. Membranes were then incubated overnight with primary α-HA antibody (3F10, Roche). The membranes were incubated with secondary antibody α-rat IRdye800 (LI-COR) 1 h. Protein signals were detected under Odyssey imaging system (LI-COR). Small RNA-seq Small RNA libraries were cloned following manufacturer’s instructions (NEBNext Multiplex Small RNA Library Prep for Illumina) and sequenced on an Illumina HiSeq1500 platform. From 20 microgram total RNA extractions, small RNAs were size selected on a 15% polyacrylamide gel electrophoresis, as described previously 18 . From BcAGO IP samples, RNA fractions were directly used for small RNA library cloning. Sequencing raw reads were demultiplexed, adapter-trimmed and quality-filtered (q = 20). Small RNA reads in the size range of 18–30 nt were considered for further analysis. Reads were mapped to the reference genome assemblies of B. cinerea strain B05.10 (Ensembl, ASM83294v1) or tomato ( S. lycopersicum accession Heintz SolGenomics Network, version SL4.0). This was performed using ShortStack3 35 with unique weighting, which uses BOWTIE with zero mismatch (-v 1) as its alignment engine. Reference sequences of rRNA, tRNA, sn/snoRNA, mRNA, repeat RNA for tomato were downloaded from the SolGenomics Network FTP site, and for B. cinerea from the Ensembl FTP site. B. cinerea retrotransposon RNA reference sequences were used, as previously annotated 34 . Small RNA read mapping against different RNA reference sequences was performed using BOWTIE2 57 . Raw read numbers were normalized to reads per million (RPM) to total read numbers mapped to the respective reference genome. Small RNA cross-kingdom target prediction was conducted with the TAPIR tool 58 using free energy ratio cut-off 0.7 and score cut-off 5.5. Furthermore, no gap, no three mismatches in a row and no two mismatches in a row in the seed region (2–12 nt) was permitted in the target alignment. Prediction of endogenous mRNA alignments for BcsRNAs was performed using GSTAr ( https://github.com/MikeAxtell/GSTAr ), which is based on RNAplex 59 . These were filtered to include only targets which are 1) from an BcsRNA that is highly expressed (> 40 locus RPM), 2) to an mRNA which is induced in a specific bcago mutant (p-value 0.58), and 3) from a BcsRNA that is either reduced in a specific bcago mutant (p-value < = 0.1, L2FC 30 RPM). BcsRNA loci annotation Small-RNA-loci were defined using a custom pipeline based on genome-wide assessment of depth, outlined in Fig. 5 B. Alignments for wildtype libraries (n = 2) were used to generate a coverage profile, normalized to RPM. A Poisson distribution was fitted to assess probability that a genomic position has expression higher than background. For this, lambda was calculated for each chromosome separately, based on 40 nucleotide windows with total depth calculated from intergenic regions, using the following formula: lambda = window_read_count / intergenic_length * intergenic_read_count Intergenic regions are defined as positions not as featuretype = mRNA in the NCBI gene annotation. Regions are then defined as positions with a Poisson probability of 10 − 4 or less and merged if they are < 150 nucleotides apart. Merged regions are trimmed to exclude edges which are < 5% of their maximum depth. Finally, regions are padded by 10 nucleotides on either edge, resulting in BcSRNA loci. Loci are assessed in terms of their basic dimensions (length, distance to prior loci) and BcsRNA profile (abundance, RPM, most common BcsRNA sequence and depth, strand preference, and complexity). Size specificity is also assessed, showing the abundance of the most common consecutive sizes for the locus. This is summarized in the field “sizecall”, which is the smallest number of consecutive sizes that are > 50% of the locus abundance, with “N” indicating loci that are not specific and likely derived from degradation. mRNA-seq Extracted total RNA was used for mRNA library cloning with prime-seq method as previously described 60 , 61 . mRNA libraries were sequenced by paired end sequencing on an Illumina HiSeq1500 platform. Raw data was demultiplexed into fastq files using the deML 62 and processed using the zUMIs pipeline (2.9.6) 63 with STAR (2.6) 64 . Transcript counts were calculated based on pseudo-alignments using the tool salmon 65 and the following command: salmon quant -l SF, with indices based on the ASM83294v1 transcriptome. Raw read counts were transformed using the DESeq2 Bioconductor package 66 . The regularized logarithm transformation (rlog) was used to transformed reads prior to visualization. Euclidean distance was used for DistHeatMap to calculate the distance within samples. Phylogenetic analysis B. cinerea AGO proteins were identified by searching the protein databases for homologies of QDE2 and SMS2 in Neurospora crassa using BLAST search 67 . The rooted phylogenetic tree was constructed with amino acid sequences of representative filamentous fungal AGO proteins in phyla of Ascomycota by RAXML method with JTT model. Fungal species and their accession number were listed in Table S1 . The alignment was performed by MAFFT and bootstrap was calculated based on 1000 replicates. The phylogenetic tree was built at CIPRES Science Gateway. Data plotting and statistical analysis GraphPad Prism 10 software was used for plotting and statistical analysis. One-way ANOVA with Tukey multiple comparisons test (p-value < 0.05) was performed for multi-samples comparison. For two samples comparison, unpaired t-test was performed. Statistical analysis was set for two-tailed p-value < 0.05 (*), p-value < 0.01 (**). Declarations Data accessibility Sequencing data have been deposited in NCBI SRA (BioProject ID PRJNA1092616). Authors` contribution AW conceptualized this work, raised funding acquisition, led project administration, supervised this work, and wrote the original draft. APC and LH performed investigation, formal analysis, data curation, wrote the original draft and developed methodologies. LO performed formal analysis and developed methodologies. AG and KS participated in investigation, formal analysis. NRJ, FS, LW and WE contributed to formal analysis and data curation. Acknowledgements We would like to thank Michael Feldbrügge and Claude Becker for critical proofreading of this work. We want to thank the Gene Center Munich for Illumina NextSeq sequencing service. 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Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14 , 417-419 (2017). Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15 , 550 (2014). Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 215 , 403-410 (1990). Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx TableS6.xlsx supplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4183067","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":285255833,"identity":"cb4406e1-119c-40ef-ad87-acd60a8cd1de","order_by":0,"name":"Arne Weiberg","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACNgYeA4bEBiCLvRlEMhPWwg/XwnOQSC2SDUAtjCDFEolEajE4wGP44OEOm3z5yIetm3kYrOWI0MK72SDxTJrlxtuJbbd5GNKNidGyTSKx7bCB4WywlsNg5+EF9gd4t/9IbPtvYDjzIFhLPUEtBgf4vzEkth0wkJdgBGtJIOyww3zFEolnkg0MeBLbbs4xSDckbMvxHsOPP3fYGci3Hz52402FtTxBW+ARYXAATBLWgADyBB00CkbBKBgFIxYAAIT2QF92Gi/3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4300-4864","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":true,"prefix":"","firstName":"Arne","middleName":"","lastName":"Weiberg","suffix":""},{"id":285255834,"identity":"3c82c483-85c3-4ac0-b609-7052d99fc1f0","order_by":1,"name":"An-Po Cheng","email":"","orcid":"https://orcid.org/0009-0004-3337-7178","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"An-Po","middleName":"","lastName":"Cheng","suffix":""},{"id":285255835,"identity":"6a786270-d551-4ba5-9792-306d803ca30a","order_by":2,"name":"Lihong Huang","email":"","orcid":"","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"Lihong","middleName":"","lastName":"Huang","suffix":""},{"id":285255836,"identity":"eb7b6f7b-ef65-47ce-82e4-f4c2df53b82a","order_by":3,"name":"Lorenz Oberkofler","email":"","orcid":"","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"Lorenz","middleName":"","lastName":"Oberkofler","suffix":""},{"id":285255837,"identity":"8221a8a1-0fe3-4f53-a4a0-712dd288efb1","order_by":4,"name":"Nathan R Johnson","email":"","orcid":"","institution":"Universidad Mayor, Santiago, Chile","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"R","lastName":"Johnson","suffix":""},{"id":285255838,"identity":"a2ff7e1d-6425-43e0-bc37-193b7e63fc0d","order_by":5,"name":"Francisco Salinas","email":"","orcid":"","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Salinas","suffix":""},{"id":285255839,"identity":"a241d193-4a76-4e0e-b719-c997d024a06a","order_by":6,"name":"Lucas Wange","email":"","orcid":"https://orcid.org/0000-0002-3275-9156","institution":"Ludwig-Maximilians Universitaet, Munich","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"","lastName":"Wange","suffix":""},{"id":285255840,"identity":"b3e79168-3c8b-47ec-8f8f-dd563a88a5c2","order_by":7,"name":"Wolfgang Enard","email":"","orcid":"https://orcid.org/0000-0002-4056-0550","institution":"Ludwig-Maximilians University","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"","lastName":"Enard","suffix":""},{"id":285255841,"identity":"cbe7ec3a-517a-4320-a73e-f259debec9e5","order_by":8,"name":"Stefan-Adrian Glodeanu","email":"","orcid":"","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"Stefan-Adrian","middleName":"","lastName":"Glodeanu","suffix":""},{"id":285255842,"identity":"bde9e994-cf03-4d5d-b6b7-ef55305ca596","order_by":9,"name":"Kyra Stillman","email":"","orcid":"","institution":"Ludwig-Maximilians University of Munich (LMU)","correspondingAuthor":false,"prefix":"","firstName":"Kyra","middleName":"","lastName":"Stillman","suffix":""}],"badges":[],"createdAt":"2024-03-28 14:50:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4183067/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4183067/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54780756,"identity":"35d982ab-dc07-4731-aa37-8b181dd0596b","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmall RNA profiling of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebcago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ko mutants.\u003c/strong\u003e A) Stem-loop RT-PCR of BcsRNAs known to induce cross-kingdom RNAi in plants. Samples were from \u003cem\u003eB. cinerea\u003c/em\u003estrains grown in axenic culture or infected tomato (Sl-infected) condition. \u003cem\u003eBcTubulin\u003c/em\u003emRNA was used as an internal control. M: 1kb DNA ladder. B) Size profiles of total BcsRNA reads detected in \u003cem\u003eB. cinerea\u003c/em\u003e WT and \u003cem\u003ebcago\u003c/em\u003e ko mutants in two biological replicates by Illumina deep sequencing. C) Schematic representation of the definition for \u003cem\u003eBcsRNA\u003c/em\u003e loci, using a \u003cem\u003eB. cinerea\u003c/em\u003eWT small RNA sequencing dataset. D) Absolute numbers of 21-22 nt size-specific \u003cem\u003eBcsRNA\u003c/em\u003eloci overlapping with different genetic annotated regions in the \u003cem\u003eB. cinerea\u003c/em\u003e genome in the context of retrotransposons (RT). E) Heat map and cluster analysis of differentially expressed \u003cem\u003eBcsRNA\u003c/em\u003e loci comparing \u003cem\u003eB. cinerea\u003c/em\u003e WT and \u003cem\u003ebcago\u003c/em\u003eko mutants.\u003c/p\u003e","description":"","filename":"Binder21.png","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/45e7ec5618a3e46144c3a6e2.png"},{"id":54780759,"identity":"7709f2bf-75d4-49ae-aa03-80052a8edf38","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":829232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBcAGO1 is required for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-induced cross-kingdom RNAi. \u003c/strong\u003eA) Fluorescence microscopy images from GFP reporter plant seedlings at different time points. A 5 ml drop of a 5 x 10\u003csup\u003e5\u003c/sup\u003e/ml conidiospore suspension was placed at the center of the leaf before placing a glass covering slip on the top that dispersed the conidiospore suspension over the entire leaf surface. The scale bars represent 1 mm. B) GFP quantification in infected seedling leaves from 0 – 42 hpi. For normalization, GFP fluorescence signal intensity at different time points (I\u003csub\u003et\u003c/sub\u003e) was subtracted with the initial GFP signal intensity (I\u003csub\u003e0\u003c/sub\u003e). C) Immunoblot analysis of GFP expression in cross-kingdom RNAi reporter plants at four time points upon \u003cem\u003eB. cinerea \u003c/em\u003einfection using a @GFP antibody. The ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) signal detected by Coomassie Brilliant Blue (CBB) staining was used as a protein loading control. Numbers indicate GFP and RuBisCo signal intensities estimated by the FIJI software. D) \u003cem\u003eB. cinerea\u003c/em\u003e genomic DNA was quantified in GFP reporter plant seedlings at 48 hpi by qRT-PCR. E) Relative mRNA expression of known tomato target genes measured at 24 hpi by qRT-PCR. F) Relative mRNA expression level of the immunity marker gene \u003cem\u003eS. lycopersicum Sl Proteinase Inhibitor (PI)-II\u003c/em\u003e.\u003cem\u003e \u003c/em\u003eIn\u003cem\u003e \u003c/em\u003eA-B), the\u003cem\u003e \u003c/em\u003etomato\u003cem\u003e SlActin\u003c/em\u003e was used as a housekeeping gene. Data points represent biological replicates and error bars indicate the standard deviation. The letters indicate significant difference using one-way ANOVA and Tukey test with p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Binder22.png","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/c39e6f0523085b7e9c3a3b48.png"},{"id":54780758,"identity":"d5fa9180-9a93-43db-a795-e3ebdcc98550","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1437694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBcAGO2 is a pathogenicity factor in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Relative mRNA expression of \u003cem\u003eBcAGO\u003c/em\u003es\u003cem\u003e \u003c/em\u003ein axenic culture or in Sl-infected samples at 48 hpi measured by qRT-PCR. \u003cem\u003eB. cinerea BcTubulin\u003c/em\u003e was used as a housekeeping gene. B) Tomato leaf infection and agar plate growth assays with \u003cem\u003eB. cinerea\u003c/em\u003e WT and different \u003cem\u003ebcago\u003c/em\u003e mutant strains. Leaf images were taken at 48 hpi to measure lesion area. Colony size on agar plates was measured in four independent cultures per strain at 3 days post cultivation start. C) Relative \u003cem\u003eB. cinerea\u003c/em\u003e genomic DNA (gDNA) was quantified in Sl-infected samples at 48 hpi by qRT-PCR, measuring tomato gDNA as a reference. In all plots, data points represent biological replicates and error bars indicate the standard deviation. The letters indicate significant difference using one-way ANOVA and Tukey test with p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Binder23.png","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/0919d8c38e8b2705dd014976.png"},{"id":54780761,"identity":"59e30ca9-6a1e-49a7-889d-0cbdc57b494c","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmall RNA binding profiles of BcAGO1 and BcAGO2 change during tomato infection.\u003c/strong\u003e A) Size distribution of total small RNA reads binding to BcAGOs in axenic culture with two biological replicates or in Sl-infected samples with three biological replicates at 48 hpi, as revealed by BcAGO IP and small RNA-seq. B) Relative fractions of BcAGO-bound total BcsRNA reads mapping to different annotated genetic loci in the \u003cem\u003eB. cinerea\u003c/em\u003e genome, including transfer RNA (tRNA), small nuclear and small nucleolar (sn/snoRNA), messenger RNA (mRNA), retrotransposons (RT). C) Normalized read counts per million (RPM) of different BcsRNAs binding to BcAGOs in axenic culture or Sl-infected samples. D) Heatmaps of the size profile for BcAGO-bound BcsRNAs mapping to defined \u003cem\u003eBcsRNA\u003c/em\u003e loci. Axenic culture and Sl-infected samples are shown on blue- and red- colour scales, respectively.\u003c/p\u003e","description":"","filename":"Binder24.png","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/e7ccb0fceb7b2841e8f1b453.png"},{"id":54780764,"identity":"8f30de2c-6e0d-4615-9e98-41f3fc406c22","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1240483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTomato small RNAs bind to BcAGOs during infection and induce cross-kingdom RNAi of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003eA) Size distribution of SlsRNAs binding to BcAGO1 or BcAGO2 in Sl-infected samples, as revealed by BcAGO IP and small RNA-seq. B) Relative BcsRNA and SlsRNA fractions binding to BcAGO1 or BcAGO2 in Sl-infected samples in three biological replicates (#). C) Left, \u003cem\u003eB. cinerea\u003c/em\u003ewas re-isolated from Sl-infected material and grown in subculture on agar plates. Subculture samples were collected from the colony edge. Right, stem-loop RT-PCR of selected SlsRNAs detected in two biological replicates (#) of re-isolated \u003cem\u003eB. cinerea \u003c/em\u003esamples collected at 20 hours or 7 days post cultivation start. Non-infected tomato leaves (\u003cem\u003eSl\u003c/em\u003e) were used as a positive control, and \u003cem\u003eB. cinerea\u003c/em\u003ethat was not re-isolated from infected tomato as well as water were used as negative controls. The SlmiR399 was used as another negative control representing a highly expressed SlsRNA which was never detected in the BcAGO IP datasets. D) Relative mRNA expression of predicted \u003cem\u003eB. cinerea\u003c/em\u003e genes targeted by SlsRNAs was measured in Sl-infected samples at 48 hpi. \u003cem\u003eB. cinerea BcActin\u003c/em\u003e was used as a housekeeping gene. E) Tomato leaf infection and agar plate growth assays with \u003cem\u003eB. cinerea\u003c/em\u003e WT and two independent target gene \u003cem\u003eBcin05g04730\u003c/em\u003e ko strains (#). Leaf images were taken at 48 hpi to measure lesion area. Colony size on agar plates was measured in six independent cultures per strain at 3-5 days post cultivation start. In all plots, data points represent biological replicates. Error bars indicate the standard deviation and numbers indicate significant difference using one-way ANOVA and the Tukey test with p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Binder25.png","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/d299f6034fbde91a76397afc.png"},{"id":63850963,"identity":"8a9a64f8-4b25-4cea-b513-b8e44a3fff6f","added_by":"auto","created_at":"2024-09-03 04:03:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4818274,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/ef5abbbf-361a-4814-ae5b-7d724cfd3c27.pdf"},{"id":54781156,"identity":"db1b4bb4-cacc-4f0a-9278-777f438d3110","added_by":"auto","created_at":"2024-04-16 16:46:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/d46ecd21ab3dd273f5d9b3ba.xlsx"},{"id":54780760,"identity":"34b4a598-ce61-422f-958b-7da294530c10","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":617387,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/c82dd2cb00da926db5773c06.xlsx"},{"id":54780767,"identity":"f0712909-2d5b-4ef2-a896-245f796032cd","added_by":"auto","created_at":"2024-04-16 16:38:15","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":816986,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/9449da2b84739d2bfee0d48e.xlsx"},{"id":54780766,"identity":"204949b7-3c14-4d49-9f28-4037812cae63","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18387,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/22c1b786f5e76a15b475209a.xlsx"},{"id":54781158,"identity":"85bc940a-989f-44f1-8cab-e94e031c4f5e","added_by":"auto","created_at":"2024-04-16 16:46:16","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2107006,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/b9d64f4f1b9186b50d001197.xlsx"},{"id":54780762,"identity":"7aa3d9f7-f45b-4133-a8fa-95b95755fdeb","added_by":"auto","created_at":"2024-04-16 16:38:14","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12367,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/4fa6a55074778b3bd0e7b666.xlsx"},{"id":54780768,"identity":"025bc7bf-4311-4f43-a666-7b023eb02dae","added_by":"auto","created_at":"2024-04-16 16:38:16","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":28554640,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4183067/v1/279b51a7fb86413940bd02e5.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fungal Argonaute proteins act in bidirectional cross-kingdom RNA interference during plant infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArgonautes (AGOs) belong to a conserved protein family and are key compounds of the RNA interference (RNAi) pathway, directing (post-) transcriptional gene silencing in animals, plants, and fungi. AGOs comprise a modular structure of PAZ, MID, and PIWI protein domains that are functional and crucial in RNA binding and gene silencing \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In plants, different AGO proteins bind to distinct classes of regulatory small RNAs, small-interfering (si)RNAs and micro(mi)RNAs, to form a ribonucleoprotein complex, known as the RNA-induced silencing complex (RISC). Previous studies revealed key biological functions of AGO proteins for the post-transcriptional regulation of RNAs and the remodeling of heterochromatin at the DNA level to fine-tune gene expression, as well as for the silencing of transposons and viruses \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn fungi, AGOs have been well characterized in non-pathogenic contexts. The \u003cem\u003eNeurospora crassa\u003c/em\u003e AGO named QDE2 is required for RNAi in the quelling process \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Another \u003cem\u003eN. crassa\u003c/em\u003e AGO, named SMS-2, mediates meiotic silencing of unpaired DNA (MSUD) during sexual reproduction \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In the basidiomycete \u003cem\u003eCryptococcus neoformans\u003c/em\u003e, sex-induced silencing (SIS) is another RNAi mechanism that depends on the fungal AGO1 \u003csup\u003e6\u003c/sup\u003e. RNAi-related heterochromatic silencing of centromere regions is dependent on AGO1 in the fission yeast \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Fungal AGO proteins contribute to transposon and transgene silencing, defense against mycovirus, endogenous gene regulation, and DNA repair \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Studies with \u003cem\u003eago\u003c/em\u003e loss-of-function mutants in diverse fungal species revealed AGO involvement in regulating metabolic processes as well as affecting fungal growth, differentiation, development, and pathogenicity \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBotrytis cinerea\u003c/em\u003e is a destructive fungal plant pathogen that infects more than 1,400 different plant species and causes the grey mold disease in many economically important crops \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eB. cinerea\u003c/em\u003e secretes small RNAs (BcsRNAs) into plant cells that bind to the plant`s own AGO1 to manipulate host immunity genes \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e; a virulence mechanism called cross-kingdom RNAi \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The biogenesis of cross-kingdom BcsRNAs requires the RNA-dependent RNA polymerase (RDR)1 and two Dicer-like (DCL) proteins \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eB. cinerea bcdcl1bcdcl2\u003c/em\u003e and \u003cem\u003ebcrdr1\u003c/em\u003e knockout mutants exhibited reduced infectivity \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCross-kingdom and trans-species RNAi have been reported in distinct pathogenic and mutualistic host-interacting organisms, including fungi, oomycetes, bacteria, parasitic plants, and nematodes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, cross-kingdom RNAi is bidirectional, because plants send small RNAs into interacting species to defend themselves against fungal pathogens \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we identified and characterized the \u003cem\u003eB. cinerea\u003c/em\u003e AGO protein family during infection of the host plant \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (tomato). Intriguingly, \u003cem\u003ebcago1\u003c/em\u003e knockout mutants failed to induce cross-kingdom RNAi similar to \u003cem\u003ebcdcl1dcl2\u003c/em\u003e and \u003cem\u003ebcrdr1\u003c/em\u003e mutants \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e; however, \u003cem\u003ebcago1\u003c/em\u003e was not impaired in virulence. Our data show that BcAGO1 mediates bidirectional cross-kingdom RNAi, from fungus to plants and \u003cem\u003evice versa\u003c/em\u003e. In contrast, BcAGO2, which is also involved in cross-kingdom RNAi, is a crucial pathogenicity factor. Lastly, we present evidence that BcAGO1, BcAGO2, and BcAGO3 control endogenous mRNA expression in \u003cem\u003eB. cinerea\u003c/em\u003e. Our study reveals the diversified regulatory functions of different BcAGOs and contributes to our understanding of the molecular mechanisms of plant infection by this economically important pathogen.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eArgonautes in the fungal plant pathogen B. cinerea\u003c/h2\u003e \u003cp\u003eIn this study, we aimed to investigate the role(s) of \u003cem\u003eB. cinerea\u003c/em\u003e BcAGOs during tomato infection. We performed a BLASTp search using the full-length protein sequence of the well-characterized \u003cem\u003eN. crassa\u003c/em\u003e QDE2 as a query in the genome sequence of the \u003cem\u003eB. cinerea\u003c/em\u003e strain B05.10 \u003csup\u003e31\u003c/sup\u003e to identify BcAGOs. Four BcAGO proteins that we termed BcAGO1, BcAGO2, BcAGO3, and BcAGO4 were predicted to comprise conserved PAZ and PIWI domains (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The MID domain and an AGO-conserved N-terminal domains were also found in the BcAGO1 and BcAGO2 using InterPro \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The PIWI domain of all four putative BcAGOs contained a conserved aspartic acid (D)/ glutamic acid (E)/D/ histidine (H) catalytic tetrad (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting that BcAGOs could function as slicer proteins \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe classified the four BcAGOs regarding functional homology by performing phylogenetic analysis including AGO amino acid sequences of different ascomycete species and the well-characterized \u003cem\u003eArabidopsis thaliana\u003c/em\u003e AtAGO1 and \u003cem\u003eHomo sapiens\u003c/em\u003e HsAGO2 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The phylogenetic tree positioned BcAGO1 into the fungal quelling clade and the BcAGO2 into the fungal MSUD clade. BcAGO3 and BcAGO4 laid outside these two clades and were of unknown homologous function.\u003c/p\u003e \u003cp\u003eThe coding gene sequence of \u003cem\u003eBcAGO4\u003c/em\u003e for the \u003cem\u003eB. cinerea\u003c/em\u003e strain B05.10 (ASM83294v1) included a predicted premature stop codon in the third exon (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA), which suggested that \u003cem\u003eBcAGO4\u003c/em\u003e is a pseudogene. The stop codon likely resulted in the automated annotation of the N- and the C-terminal part of \u003cem\u003eBcAGO4\u003c/em\u003e into two separate genes, \u003cem\u003eBcin15g05050\u003c/em\u003e and \u003cem\u003eBc15g05060\u003c/em\u003e, as deposited in the Ensembl database. This point mutation was not found in genome sequences of two other released \u003cem\u003eB. cinerea\u003c/em\u003e genome sequences of the strains T4 (GCA_000227075) and BcDW1 (GCA_000349525), respectively (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). To exclude the possibility that this mutation was an error introduced during whole genome sequencing, we cloned \u003cem\u003eBcAGO4\u003c/em\u003e from the B05.10 strain and confirmed this mutation being present by Sanger sequencing (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). This shows that there is within-species genetic variation at the \u003cem\u003eBcAgo4\u003c/em\u003e locus among \u003cem\u003eB. cinerea\u003c/em\u003e strains. In the following experiments of this study, we used the strain B05.10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAccumulation of fungal cross-kingdom small RNAs is controlled by BcAGO1\u003c/h2\u003e \u003cp\u003eWe first explored the possibility that BcAGOs might control BcsRNA accumulation. For this, we generated \u003cem\u003ebcago\u003c/em\u003e ko mutants and isolated two single knockout (ko) mutant strains for each BcAGO, referred to as \u003cem\u003ebcago1\u003c/em\u003e, \u003cem\u003ebcago2\u003c/em\u003e, \u003cem\u003ebcago3\u003c/em\u003e, \u003cem\u003ebcago4\u003c/em\u003e (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). We performed stem-loop reverse transcription (RT) PCR of BcsRNAs, collecting samples of \u003cem\u003eB. cinerea\u003c/em\u003e grown in axenic culture and from infected tomato leaves (Sl-infected). We chose the BcsRNA3.1, BcsRNA3.2, BcsRNA20, because these were previously found to induce cross-kingdom RNAi of tomato genes during \u003cem\u003eB. cinerea\u003c/em\u003e infection \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We determined that \u003cem\u003ebcago1\u003c/em\u003e ko mutants and a \u003cem\u003ebcago1ago2\u003c/em\u003e double-ko (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA) lost the accumulation of BcsRNA3.1, BcsRNA3.2, and BcsRNA20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). BcsRNA accumulation was reconstituted in a 3xHA-tagged BcAGO1 complementation strain (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB, Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next performed comparative small RNA deep sequencing analysis to profile global changes of BcsRNA accumulation. Two independent strains of each \u003cem\u003ebcago\u003c/em\u003e ko mutant and \u003cem\u003eB. cinerea\u003c/em\u003e wild type (WT) were grown in axenic culture for small RNA extraction and Illumina library preparation. Upon raw read processing, we mapped reads to the \u003cem\u003eB. cinerea\u003c/em\u003e reference genome allowing a maximum of one mismatch. These BcsRNAs mapped either one or multiple times, when we chose a single \u0026ldquo;best\u0026rdquo; map-location with multiple-mappers \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA). BcsRNA reads mapped to various annotated genomic regions (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB) and showed an overall size enrichment for 21\u0026ndash;22 nt reads (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). An exception was \u003cem\u003ebcago1\u003c/em\u003e, which lost most of the 21\u0026ndash;22 nt BcsRNAs that were mainly derived from retrotransposons (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo be able to measure quantitative differences in BcsRNA accumulation, we annotated for the first time \u003cem\u003eBcsRNA\u003c/em\u003e producing genomic loci in \u003cem\u003eB. cinerea\u003c/em\u003e, using one of the \u003cem\u003eB. cinerea\u003c/em\u003e WT small RNA-seq datasets. We defined \u003cem\u003eBcsRNA\u003c/em\u003e loci as regions with coverage above the chromosomal average, defined by a Poisson probability P\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e. Nearby regions (\u0026lt;\u0026thinsp;150 bases apart), given a 10-nucleotide buffer on both sides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eC). With these criteria, we identified in total 4,397 \u003cem\u003eBcsRNA\u003c/em\u003e loci (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), covering 96.3% of the mapped BcsRNAs (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD). Annotated loci include many which were not size-specific and are likely derived from but not limited to mRNA degradation (Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eE). Most of the 21\u0026ndash;22 nt size-specific \u003cem\u003eBcsRNA\u003c/em\u003e loci were found in non-annotated genomic regions, nearby genes, and within gene bodies of exonic and intronic regions. More than 50% of these 21\u0026ndash;22 nt size-specific \u003cem\u003eBcsRNA\u003c/em\u003e loci overlapped with retrotransposons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). As non-size-specific BcsRNAs derived from mRNAs were likely degradation products and not BcAGO-associated, these served as controls for normalization of size-specific BcsRNAs (Figure S8A-B). All differentially expressed \u003cem\u003eBcsRNA\u003c/em\u003e loci identified by pairwise comparison between \u003cem\u003ebcago\u003c/em\u003e ko mutants and \u003cem\u003eB. cinerea\u003c/em\u003e WT are listed in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. Normalized read counts of \u003cem\u003eBcsRNA\u003c/em\u003e loci confirmed that \u003cem\u003ebcago1\u003c/em\u003e ko mutants showed the strongest change in BcsRNA expression, with mostly decreased 21\u0026ndash;22 nt size-specific \u003cem\u003eBcsRNA\u003c/em\u003e loci expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Figure S8C). Therefore, \u003cem\u003ebcago1\u003c/em\u003e exhibited reduced cross-kingdom small RNA accumulation, similar to \u003cem\u003ebcrdr1\u003c/em\u003e and \u003cem\u003ebcdcl1dcl2\u003c/em\u003e double-ko mutants, as reported in previous studies \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBcAGO1 is required for fungal-induced cross-kingdom RNAi\u003c/h2\u003e \u003cp\u003eSince \u003cem\u003ebcago1\u003c/em\u003e ko mutants lost accumulation of 21\u0026ndash;22 nt BcsRNAs, we anticipated that \u003cem\u003ebcago1\u003c/em\u003e ko mutant might be compromised in cross-kingdom RNAi during plant infection. We recently developed a GFP \u0026ldquo;switch on\u0026rdquo; cross-kingdom RNAi reporter system carrying the BcsRNA3.1 and BcsRNA3.2 target sites of \u003cem\u003eA. thaliana\u003c/em\u003e genes. When infecting transgenic \u003cem\u003eA. thaliana\u003c/em\u003e reporter plants with \u003cem\u003eB. cinerea\u003c/em\u003e, GFP expression is turned on within 24\u0026ndash;48 hpi \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We used this \u003cem\u003eA. thaliana\u003c/em\u003e reporter line to further inspect cross-kingdom RNAi with the \u003cem\u003ebcago1\u003c/em\u003e and \u003cem\u003ebcago2\u003c/em\u003e ko mutants. We infected seedlings with \u003cem\u003eB. cinerea\u003c/em\u003e WT, \u003cem\u003ebcago1\u003c/em\u003e, and \u003cem\u003ebcago2\u003c/em\u003e and recorded GFP signal expression in infected leaves in a time course by fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This analysis indicated some background fluorescence intensity (I\u003csub\u003e0\u003c/sub\u003e) in the reporter plants. Therefore, we calculated relative increase of GFP fluorescence intensity (I\u003csub\u003et\u003c/sub\u003e-I\u003csub\u003e0\u003c/sub\u003e) at different time points of infection, following a previously reported analysis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Inoculation with \u003cem\u003ebcago1\u003c/em\u003e ko strain did not induce GFP signal at any measured time point, while \u003cem\u003ebcago2\u003c/em\u003e caused an increase in GFP signal intensity, but to a lesser extent than \u003cem\u003eB. cinerea\u003c/em\u003e WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These observations were corroborated in an immunoblot analysis of GFP protein levels of infected leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Figure S9). We measured \u003cem\u003eB. cinerea\u003c/em\u003e genomic DNA levels in the \u003cem\u003eA. thaliana\u003c/em\u003e reporter plants at the same time point as the GFP measurements and observed no difference between WT, \u003cem\u003ebcago1\u003c/em\u003e, or \u003cem\u003ebcago2\u003c/em\u003e inoculations (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This suggested that the observed lower GFP activation was not due to a diminished \u003cem\u003eB. cinerea\u003c/em\u003e colonization. Based on the observation, we concluded that BcAGO1 was required for and BcAGO2 also contributed to cross-kingdom RNAi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next measured mRNA levels of the known tomato BcsRNA3.1, BcsRNA3.2, and BcsRNA20 target genes, namely \u003cem\u003eSlVPS, SlMPKKK4\u003c/em\u003e, and \u003cem\u003eSlBhlh63\u003c/em\u003e \u003csup\u003e21, 34\u003c/sup\u003e. \u003cem\u003eSlMPKKK4\u003c/em\u003e and \u003cem\u003eSlBhlh63\u003c/em\u003e were no longer suppressed upon infection with \u003cem\u003ebcago1\u003c/em\u003e ko strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), agreeing with the loss of BcsRNA3.2 and BcsRNA20 accumulation and previous findings that the \u003cem\u003ebcrdr1\u003c/em\u003e also lost BcsRNA accumulation and showed no longer suppression of these target genes upon infection \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Target gene suppression was reconstituted upon infection with a 3xHA-BcAGO1 complementation strain. \u003cem\u003eSlMPKKK4\u003c/em\u003e and \u003cem\u003eSlBhlh63\u003c/em\u003e showed similar expression levels upon infection with \u003cem\u003ebcago2\u003c/em\u003e and \u003cem\u003eB. cinerea\u003c/em\u003e WT. \u003cem\u003eSlVPS\u003c/em\u003e expression was suppressed upon infection with \u003cem\u003ebcago1\u003c/em\u003e at similar levels as \u003cem\u003eB. cinerea\u003c/em\u003e WT, but \u003cem\u003eSlVPS\u003c/em\u003e was not suppressed upon infection with \u003cem\u003ebcago2\u003c/em\u003e, indicating that this gene was suppressed by a BcAGO2-dependent BcsRNA. All three tomato target genes were no longer suppressed upon infection with a \u003cem\u003ebcago1ago2\u003c/em\u003e double-ko. These results confirmed that both, BcAGO1 and BcAGO2, might be involved in cross-kingdom RNAi, but act on different plant target genes. The tomato immunity marker gene \u003cem\u003eSl-Proteinase Inhibitor (PI)-II\u003c/em\u003e was used as a non-target gene control. \u003cem\u003eSl-PI-II\u003c/em\u003e was more strongly induced when infecting with \u003cem\u003ebcago2\u003c/em\u003e ko mutants compared to \u003cem\u003eB. cinerea\u003c/em\u003e WT or \u003cem\u003ebcago1\u003c/em\u003e ko (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). MPKKK4 is part of a conserved plant immune signaling cassette that involves the downstream targets MPK3/MPK6 and WRKY33 \u003csup\u003e36\u003c/sup\u003e. When infecting tomato with \u003cem\u003ebcago1\u003c/em\u003e ko mutants, \u003cem\u003eSlMPK3\u003c/em\u003e and \u003cem\u003eSlWRKY33\u003c/em\u003e were higher expressed compared to infection with \u003cem\u003eB. cinerea\u003c/em\u003e WT or \u003cem\u003ebcago2\u003c/em\u003e (Figure S10A). Accordingly, infected tomato revealed higher ROS accumulation upon \u003cem\u003ebcago1\u003c/em\u003e infection compared to \u003cem\u003eB. cinerea\u003c/em\u003e WT or \u003cem\u003ebcago2\u003c/em\u003e ko (Figure S10B). Thus, \u003cem\u003ebcago1\u003c/em\u003e and \u003cem\u003ebcago2\u003c/em\u003e mutants were hampered in host immune suppression, but of different immunity pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eB. cinerea BcAGO2 is a fungal pathogenicity factor\u003c/h2\u003e \u003cp\u003eTo gain further information on the functional role of BcAGOs during tomato infection, we measured \u003cem\u003eBcAGO\u003c/em\u003e mRNA levels in axenic culture and in Sl-infected sample conditions by quantitative reverse transcription PCR (qRT-PCR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). \u003cem\u003eBcAGO1\u003c/em\u003e was more highly expressed under both conditions compared to \u003cem\u003eBcAGO2\u003c/em\u003e, \u003cem\u003eBcAGO3\u003c/em\u003e, and \u003cem\u003eBcAGO4\u003c/em\u003e. The \u003cem\u003eBcAGO2\u003c/em\u003e and \u003cem\u003eBcAGO3\u003c/em\u003e displayed up-regulation at 1 day post inoculation (dpi). \u003cem\u003eBcAGO4\u003c/em\u003e showed low expression in both conditions. We next compared disease severity induced by \u003cem\u003ebcago\u003c/em\u003e ko mutants using a tomato leaf infection assay. Using conidiospore drop (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) or agar plug inoculation methods (Figure S11), we observed that \u003cem\u003ebcago2\u003c/em\u003e ko mutants induced smaller lesion areas compared to WT. The \u003cem\u003ebcago1ago2\u003c/em\u003e double-ko showed reduced virulence, similar to \u003cem\u003ebcago2\u003c/em\u003e single ko. Conversely, reduced pathogenicity of \u003cem\u003ebcago2\u003c/em\u003e was reverted in 3xHA-\u003cem\u003eBcAGO2\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Figure S11A). Accordingly, we measured significantly less \u003cem\u003eB. cinerea\u003c/em\u003e genomic DNA, a proxy for pathogen biomass, in leaf tissue infected with the \u003cem\u003ebcago2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). When growing \u003cem\u003ebcago\u003c/em\u003e ko mutants on agar plates, we observed normal fungal growth and development in all strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Figure S12).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these results, we concluded that BcAGO2 is a pathogenicity factor in \u003cem\u003eB. cinerea\u003c/em\u003e and hampered cross-kingdom RNAi could explain the reduced pathogenicity. Surprisingly, \u003cem\u003ebcago1\u003c/em\u003e ko mutants did not display any noticeable change in tomato infection, although \u003cem\u003ebcago1\u003c/em\u003e was no longer inducing cross-kingdom RNAi and was hampered in suppressing tomato immunity. Thus, we speculated that BcAGO1 must have a second function during plant infection, and this could be to bind plant-derived small RNAs that could trigger silencing of fungal genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBcAGO1 facilitates tomato-induced cross-kingdom RNAi\u003c/h2\u003e \u003cp\u003eTomato small RNAs might be delivered during infection into \u003cem\u003eB. cinerea\u003c/em\u003e cells and hijacked BcAGO1 to suppress fungal pathogenicity genes as a defense response. A previous report showed that \u003cem\u003eA. thaliana\u003c/em\u003e delivers small RNAs into \u003cem\u003eB. cinerea\u003c/em\u003e to silence pathogenicity-related genes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, but a role of BcAGOs was not explored. To identify tomato small RNAs that bind to BcAGOs during infection, we performed BcAGO co-immunopurification (BcAGO IP) coupled to small RNA deep sequencing. For this experiment, we used transgenic \u003cem\u003eB. cinerea\u003c/em\u003e strains expressing 3xHA-tagged BcAGO1 or 3xHA-BcAGO2 in the \u003cem\u003ebcago1\u003c/em\u003e or \u003cem\u003ebcago2\u003c/em\u003e ko mutant background, respectively. As observed previously in this study, the transgenic 3xHA-BcAGO1 strains could revert the loss of BcsRNA accumulation in the \u003cem\u003ebcago1\u003c/em\u003e and 3xHA-BcAGO2 reverted the reduced disease phenotype of the \u003cem\u003ebcago2\u003c/em\u003e ko strains, confirming that both 3xHA-BcAGO constructs were functional. Upon immunoblot confirmation of successful BcAGO IP (Figure S13), we isolated small RNAs for cloning and Illumina-based sequencing. We sequenced two biological replicates from axenic culture and three biological replicates for Sl-infected samples (Figure S14A). Small RNA sequencing analysis revealed a shift of size enrichment from 24 nt reads in axenic culture to 21\u0026ndash;22 nt reads in Sl-infected samples for both BcAGO1 and BcAGO2 IPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When mapping reads to the \u003cem\u003eB. cinerea\u003c/em\u003e or the tomato reference genomes, we identified sequences that exclusively mapped to one or the other species with at least one mismatch to the opposite reference genome, accordingly. For BcsRNAs, 21\u0026ndash;22 nt long retrotransposon-derived reads were enriched in Sl-infected samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The cross-kingdom BcsRNA3.1, BcsRNA3.2, BcsRNA5, and BcsRNA20, which are all derived from retrotransposons \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, indicated higher read numbers in Sl-infected samples compared to axenic culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This result was consistent with a previous finding that retrotransposon-derived 21\u0026ndash;22 nt BcsRNAs are induced upon plant infection \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Likewise, when mapping reads to our defined \u003cem\u003eBcsRNA\u003c/em\u003e loci, we observed overlapping BcsRNA accumulation between BcAGO1 and BcAGO2 (Figure S14B-D) and a size preference of 21\u0026ndash;22 nt long reads in size-specific loci (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong BcAGO-bound tomato small RNAs (SlsRNAs), 21 nt long reads were enriched in the BcAGO1 and BcAGO2 IPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Remarkably, SlsRNAs reached up to 50% of mapped read counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating massive invasion of tomato small RNAs into BcAGOs during infection. The SlsRNA reads mapped to different annotated genetic loci, with the vast majority of them being associated with DNA repeats, mRNAs, tRNAs, and rRNAs (Figure S15). Similar to the BcAGO-bound BcsRNA fraction, the SlsRNAs largely overlapped in binding to BcAGO1 and BcAGO2 during infection (Figure S14D). Among most abundant SlsRNAs, higher read numbers were counted in BcAGO1 IP samples compared to BcAGO2 IP (Figure S16A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next investigated the possibility that SlsRNA candidates silence \u003cem\u003eB. cinerea\u003c/em\u003e genes during infection. We considered 21\u0026ndash;22 nt SlsRNA reads with an average of \u0026gt;\u0026thinsp;100 RPM in the BcAGO1 or BcAGO2 IP small RNA-seq datasets. We filtered out all SlsRNA reads mapping to ribosomal RNA (rRNA), small nuclear or nucleolar RNA (snRNA/snoRNA), or protein coding transcripts (in sense orientation), as these did not likely represent regulatory RNAs but RNA degradation products. In total, we predicted 74 \u003cem\u003eB. cinerea\u003c/em\u003e target genes of 21 SlsRNA candidates using the TAPIR tool with stringent parameter setting (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). These SlsRNA candidates were detected in all BcAGO IP datasets (Figure S16A). We chose a subset of 10 SlsRNAs to confirm that these invaded into \u003cem\u003eB. cinerea\u003c/em\u003e during tomato infection. For this, we re-isolated \u003cem\u003eB. cinerea\u003c/em\u003e from Sl-infected leaf tissue to subculture on agar plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). \u003cem\u003eB. cinerea\u003c/em\u003e subculture samples were taken from the colony edge after 20 h or 7 d for SlsRNA analysis. The SlmiRNA159, SlmiR162, SlsRNA4, SlsRNA11, and SlsRNA12 were detected in independent \u003cem\u003eB. cinerea\u003c/em\u003e re-isolation samples by stem-loop RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Figure S16B). These SlsRNAs were not detected in \u003cem\u003eB. cinerea\u003c/em\u003e mycelium that was not re-isolated from infected tomato but were present in tomato samples. SlsRNAs were also detected in re-isolated \u003cem\u003ebcago1\u003c/em\u003e, \u003cem\u003ebcago2\u003c/em\u003e and \u003cem\u003ebcago1ago2\u003c/em\u003e ko mutant strains, as well as in a \u003cem\u003ebcrdr1\u003c/em\u003e ko mutant strain that was recently characterized to be required for BcsRNA accumulation and cross-kingdom RNAi \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e (Figure S16C). In this RT-PCR assay, the SlmiR399 served as a negative control SlsRNA, because it was not detected in the BcAGO IP small RNA datasets of this work.\u003c/p\u003e \u003cp\u003eTo further analyze if SlsRNAs could suppress \u003cem\u003eB. cinerea\u003c/em\u003e target genes through BcAGOs during infection, we measured mRNA expression of predicted target genes in Sl-infected samples, comparing \u003cem\u003eB. cinerea\u003c/em\u003e WT with \u003cem\u003ebcago1\u003c/em\u003e, \u003cem\u003ebcago2\u003c/em\u003e, and \u003cem\u003ebcago1ago2\u003c/em\u003e ko strains. Among 14 tested \u003cem\u003eB. cinerea\u003c/em\u003e candidate genes, \u003cem\u003eBcin12g05240\u003c/em\u003e, \u003cem\u003eBcin05g04730\u003c/em\u003e, \u003cem\u003eBcin07g06910\u003c/em\u003e, and \u003cem\u003eBcin04g05820\u003c/em\u003e displayed higher expression in \u003cem\u003ebcago1\u003c/em\u003e and \u003cem\u003ebcago1ago2\u003c/em\u003e ko mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Figure S17A). Failed mRNA suppression was not due to \u003cem\u003ebcago1\u003c/em\u003e loss-of-function \u003cem\u003eper se\u003c/em\u003e, because elevated mRNA expression was not evident when comparing \u003cem\u003eB. cinerea\u003c/em\u003e WT and \u003cem\u003ebcago1\u003c/em\u003e ko strains grown in axenic culture (Figure S17B). Moreover, \u003cem\u003eBcin05g04730\u003c/em\u003e was transcriptionally upregulated in Sl-infected samples upon \u003cem\u003eB. cinerea\u003c/em\u003e WT infection, indicating that this gene might be relevant for fungal infection (Figure S17C). The \u003cem\u003eBcin05g04730\u003c/em\u003e encodes for a putative Serine/Threonine Protein kinase. Hence, we chose \u003cem\u003eBcin05g04730\u003c/em\u003e for targeted gene ko to assess its potential role in tomato infection. Two independent ko strains (Figure S18) displayed reduced lesion area induction when infecting tomato and strains grew more slowly on agar plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Thus, we uncovered a novel function of \u003cem\u003eBcin05g04730\u003c/em\u003e in fungal pathogenicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eB. cinerea BcAGOs regulate fungal mRNAs related to plant infection\u003c/h2\u003e \u003cp\u003eApart from cross-kingdom RNAi, BcAGOs could regulate expression of endogenous fungal genes related to pathogenicity. To identify such genes, we conducted a mRNA-seq experiment comparing \u003cem\u003eB. cinerea\u003c/em\u003e WT and \u003cem\u003ebcago\u003c/em\u003e ko mutant strains grown in axenic culture. PCA of all mapped read counts verified overall sufficient reproducibility of datasets (Figure S19A). Pairwise comparison of \u003cem\u003eB. cinerea\u003c/em\u003e WT versus \u003cem\u003ebcago\u003c/em\u003e ko mutants revealed differentially expressed genes (DEGs) using cut-off criteria of one-half fold-change expression (log fold change\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;0.58) and adjusted p-value of 0.1 (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). In the \u003cem\u003ebcago1\u003c/em\u003e ko mutant, many up-or down-regulated genes were annotated to have extracellular enzymatic activities and comprised known or predicted virulence factors (Figure S20A). In the \u003cem\u003ebcago2\u003c/em\u003e background, we confirmed that \u003cem\u003eBcin02g00620\u003c/em\u003e, \u003cem\u003eBcin02g01230\u003c/em\u003e, and \u003cem\u003eBcin09g00460\u003c/em\u003e were also differentially expressed during tomato infection (Figure S20B) indicating putative functions in pathogenicity. The largest number of DEGs (337 up- and 400 down) were found in the \u003cem\u003ebcago3\u003c/em\u003e ko mutant (Figure S19B) which included many genes belonging to the phytotoxins Botrydial and Botcinic acid biosynthetic gene clusters (Figure S20C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, in this study, we discovered complex RNA regulation executed by different BcAGOs during host infection in at least three different functions: i) BcAGO1 is required for fungal induced cross-kingdom RNAi by controlling BcsRNA accumulation, ii) BcAGO1 binds SlsRNAs that suppress fungal pathogenicity genes, iii) BcAGO1, BcAGO2, and BcAGO3 regulate endogenous mRNA expression including fungal pathogenicity genes (Figure S21).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCross-kingdom RNAi is an emerging field in host-pathogen interaction research and bidirectional cross-kingdom RNAi has been described in fungal-plant interaction. In one direction, \u003cem\u003eB. cinerea\u003c/em\u003e secretes BcsRNAs into its host plants tomato and \u003cem\u003eA. thaliana\u003c/em\u003e that bind to the plants\u0026acute; own AGO1 to silence host immunity genes \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In the counter-direction, \u003cem\u003eA. thaliana\u003c/em\u003e secretes small RNAs that can trigger gene suppression in \u003cem\u003eB. cinerea\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The role of pathogen AGOs in cross-kingdom RNAi had not been explored. We herewith provide new mechanistic insights of the diversified regulatory functions of different BcAGO family members in bidirectional cross-kingdom RNAi and in plant infection.\u003c/p\u003e \u003cp\u003eProfiling the small RNA transcriptome revealed that BcAGO1 is required for the accumulation of cross-kingdom BcsRNAs. Reduction but not complete loss of small RNA has been reported in \u003cem\u003eago\u003c/em\u003e loss-of-function mutants before \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It is argued that small RNAs bound to AGO could experience transient protection against rapid nuclease degradation. Alternatively, BcAGO1 might regulate BcsRNA biogenesis. For instance, the fungal \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e MoAGO2 interferes with RNAi triggered by hairpin and retrotransposon-derived small RNAs through the MoAGO1 and MoAGO3 \u003csup\u003e39\u003c/sup\u003e. A similar BcAGO1-mediated RNAi feedback loop might also exist in \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWhen using a GFP switch-on reporter assay \u003cem\u003ein planta\u003c/em\u003e, which allowed us to measure fungal-induced cross-kingdom RNAi in plants over a time course of infection, the \u003cem\u003ebcago1\u003c/em\u003e ko mutant failed in reporter activation. This is in agreement with a previous finding that \u003cem\u003eB. cinerea bcrdr1\u003c/em\u003e and \u003cem\u003ebcdcl1bcdcl2\u003c/em\u003e ko strains, which were impaired in cross-kingdom BcsRNA production \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e also failed to induce cross-kingdom RNAi in the same reporter plant \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Using this assay, we also observed that the \u003cem\u003ebcago2\u003c/em\u003e ko mutant was compromised in cross-kingdom RNAi. However, BcsRNA accumulation was largely unaltered in the \u003cem\u003ebcago2\u003c/em\u003e, implying another role of BcAGO2 in cross-kingdom RNAi, compared to BcAGO1, BcDCLs, and BcRDR1. For instance, \u003cem\u003eA. thaliana\u003c/em\u003e AGO1 participates in packaging small RNA into extracellular vesicles (EVs) for secretion \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. EVs are also produced by \u003cem\u003eB. cinerea\u003c/em\u003e that contain BcsRNAs and are taken up into \u003cem\u003eA. thaliana\u003c/em\u003e cells via clathrin-mediated endocytosis \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Therefore, BcAGO2 might play a role in BcsRNA sorting for secretion, which could explain compromised cross-kingdom RNAi when infecting reporter plants with \u003cem\u003ebcago2\u003c/em\u003e ko.\u003c/p\u003e \u003cp\u003eUsing \u003cem\u003ebcago\u003c/em\u003e ko mutants in a tomato leaf infection assay, we observed reduced pathogenicity in the \u003cem\u003ebcago2\u003c/em\u003e mutant. Likewise, single \u003cem\u003eago\u003c/em\u003e gene ko in the apple canker fungus \u003cem\u003eValsa mali\u003c/em\u003e reduced virulence \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. An \u003cem\u003eago\u003c/em\u003e ko in the wheat-infecting fungus \u003cem\u003eZymoseptoria tritici\u003c/em\u003e led to stop the production of asexual propagules \u003cem\u003ein planta\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. A possible reason for the reduced pathogenicity in \u003cem\u003ebcago2\u003c/em\u003e could be the limited capability to induce cross-kingdom RNAi, but it would be necessary to further explore this causality in detail.\u003c/p\u003e \u003cp\u003eCross-kingdom RNAi was reported in both directions during \u003cem\u003eB. cinerea\u003c/em\u003e-plant interaction \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We here found evidence that BcAGO1 was exploited for tomato-induced cross-kingdom RNAi. By profiling the BcAGO-bound small RNA repertoire during tomato infection, we identified at least 21 tomato SlsRNAs that were predicted to target 74 \u003cem\u003eB. cinerea\u003c/em\u003e mRNAs. We demonstrated that four \u003cem\u003eB. cinerea\u003c/em\u003e genes were suppressed during infection in a BcAGO1-dependent manner and proofed that one target gene, a serine/threonine protein kinase, was part of \u003cem\u003eB. cinerea\u003c/em\u003e pathogenicity. Therefore, studying pathogen AGO-associated host small RNAs during infection, not only deciphered tomato small RNAs that were capable to induce cross-kingdom RNAi, but also promise to reveal novel \u003cem\u003eB. cinerea\u003c/em\u003e pathogenicity factors. Interestingly, protein kinases were also previously identified as cross-kingdom RNAi targets of \u003cem\u003eB. cinerea\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and the oomycete \u003cem\u003eHyaloperonospora arabidopsidis\u003c/em\u003e small RNAs \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e and tomato host plant species, revealing that manipulating this class of enzymes has evolved in diverse biotic interactions.\u003c/p\u003e \u003cp\u003eDifferential mRNA transcriptome analysis suggests that BcAGOs regulate endogenous fungal genes related to pathogenicity. As an example, up-regulation of a predicted \u003cem\u003eMFS transporter\u003c/em\u003e (\u003cem\u003eBcin02g00620\u003c/em\u003e) during tomato infection is dependent on BcAGO2. It is known that MFS transporters provide resistance to various fungitoxic compounds and regulate \u003cem\u003eB. cinerea\u003c/em\u003e tolerance to glucosinolate-breakdown products, which is required for pathogenicity \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. BcAGO3 regulates expression of several phyotoxin biosynthesis genes belonging to the Botrydial (BOT) and Botcinic acid (BOA) gene clusters \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Previous findings also showed that AGO and other RNAi components in the fungal pathogen \u003cem\u003eF. graminearum\u003c/em\u003e are required for mycotoxin deoxynivalenol production and full virulence \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, suggesting that regulatory function of fungal AGOs in toxin production is conserved.\u003c/p\u003e \u003cp\u003eIn summary, we uncovered diversified functions of BcAGOs during plant infection, including regulation of fungal endogenous genes as well as fungal- and plant-induced cross-kingdom RNAi. This led to new mechanistic insights into the complex regulatory roles of pathogen AGOs during plant infection. Translating such knowledge into innovative siRNA tools promise to improve RNAi-based crop protection strategies in future.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eFungal and plant materials\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e Pers. Fr. (\u003cem\u003eBotryotinia fuckeliana\u003c/em\u003e [de Bary] Whetzel) strain B05.10 \u003csup\u003e46\u003c/sup\u003e was used in the study and was cultured in complete HA media \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, if not otherwise notified. \u003cem\u003eB. cinerea\u003c/em\u003e strains were cultivated at 20\u0026deg;C under constant irradiation for conidiation or under constant dark condition. Sporulated mycelia of \u003cem\u003eB. cinerea\u003c/em\u003e were eluted with distilled water and filtered by Miracloth (Merck Millipore) to collect conidiospore suspension. Conidia were stored in 25% glycerol and kept at -80\u0026deg;C for a long-term storage. Mycelial plugs (\u0026Oslash;=0.4 cm) were collected in distilled water at 4\u0026deg;C for a temporary storage. Mycelia collected from Sl-infected leaves were stored for inoculation assay to activate fungal virulent genes.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eSolanum lycopersicum\u003c/em\u003e (cultivar Heintz 1706) was grown in a climate chamber under controlled condition (16 h light/8 h dark, 24\u0026deg;C, 60% relative humidity). \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Columbia (Col)-0 was grown under short-day conditions (8 h light/16 h dark, 22\u0026deg;C, 60% relative humidity).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eFungal transformation\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eBotrytis\u003c/em\u003e transformation was performed as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e with minor modifications. \u003cem\u003eBotrytis\u003c/em\u003e protoplasts were mixed with SH agar (0.6 M sucrose, 5 mM Tris-HCl (pH 6.5), 1 mM (NH\u003csub\u003e4\u003c/sub\u003e)H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 8 g/L agar) without any antibiotics and incubated in darkness for 24 h after transformation. Another layer of SH agar containing hygromycin B (50 \u0026micro;g/ml) or nourseothricin (50 \u0026micro;g/ml) was added to the top after pre-incubation. The plates were kept in darkness for 3\u0026ndash;5 days until single colony isolation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eFungal growth assay\u003c/h2\u003e\n \u003cp\u003eFor observation of the growth rate of \u003cem\u003eBotrytis\u003c/em\u003e wild-type and transgenic strains, a droplet of a suspension (20 \u0026micro;l, 5x10\u003csup\u003e4\u003c/sup\u003e spores/ml) with spores in distilled water was pipetted on HA agar media. Petri dishes were incubated for 5 days at room temperature. Mycelial growth was determined by measuring the radial growth of colonies.\u003c/p\u003e\n \u003cp\u003eConidia were collected from HA media culturing for two weeks under constant light to observe the conidial shape of \u003cem\u003eBotrytis\u003c/em\u003e wild-type and transgenic strains. Conidium width and length were measured from over 200 spores for each transformant.\u003c/p\u003e\n \u003cp\u003eConidia were eluted in distilled water by stirring sporulated mycelium on media plates. The number of conidia produced was determined by counting the spores microscopically with the hemocytometer (Neubauer improved, Marienfeld). Each genotype had six biological replicates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eInfection assays\u003c/h2\u003e\n \u003cp\u003eFor infection assay with spores, conidia were eluted from sporulated \u003cem\u003eB. cinerea\u003c/em\u003e with 1% malt extract \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e suspension buffer. A droplet of 20 \u0026micro;l (5x10\u003csup\u003e4\u003c/sup\u003e spores/ml) conidial suspension were inoculated on detached leaves of six weeks old tomato plants. Inoculation assay with mycelial plugs (\u0026Oslash;=0.4 cm) was performed according to the previous method \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Mycelia on agar plates of \u003cem\u003eB. cinerea\u003c/em\u003e were inoculated on detached leaves of four weeks old tomato plants. The lesion area was measured using Fiji software (ImageJ version 2.1.0/1.53c). For quantifying mycelial growth of \u003cem\u003eB. cinerea\u003c/em\u003e, leaf discs three infected leaves were collected for genomic DNA extraction and qRT-PCR by using SYBR Green (Thermo Scientific) with qPCR cycler (CFX96, Bio-Rad).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eReactive oxygen species (ROS) measurements\u003c/h2\u003e\n \u003cp\u003eROS detection was performed by luminol-based assay as previous study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Infected tomato leaf discs were collected at 12 hpi and incubated in 96-well plates with 200 \u0026micro;l of sterile water for 12 h in the dark. The water was replaced with 100 \u0026micro;l of working solution (200 \u0026micro;M luminol L-012, 10 \u0026micro;g/ml horseradish peroxidase). 100 nM Flg22 was used as positive control. Luminescence was detected using Photek camera over an hour. Plots are averages of six or eight leaf disks from independent leaves for two independent replicates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eDNA and RNA extraction\u003c/h2\u003e\n \u003cp\u003eGenomic DNA of pure mycelium or sporulated mycelium was isolated from at least 30 transformants for each construct using CTAB according to the previous method \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e prior to chloroform/isoamyl alcohol extraction and isopropanol precipitation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e with minor modifications \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Mycelia growing on HA media dishes were harvested after cultivation for five to seven days in constant light or overlayed with cellophane for three days under constant dark condition. Six pairs of primers were used for genotyping transgenic transformants for each genotype of \u003cem\u003eB. cinerea\u003c/em\u003e. GoTaq G2 DNA Polymerase (Promega) was used for genotyping with cycler. Primer oligos used for genotyping were listed in Table \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eRT-PCR\u003c/h2\u003e\n \u003cp\u003eSix-week-old tomatoes were treated with conidial suspension (2x10\u003csup\u003e5\u003c/sup\u003e spores/ml) of \u003cem\u003eBotrytis\u003c/em\u003e wild-type or ko mutant strains using a versatile sprayer (Roth Labware \u0026reg;). Tomato or \u003cem\u003eA. thaliana\u003c/em\u003e leaf discs were collected after 24, 48, 72 hours of treatment for \u003cem\u003eAGO\u003c/em\u003e gene expression analysis. Four leaf discs (\u0026Oslash;=0.4 cm) were collected as one biological replicate. \u003cem\u003eBotrytis\u003c/em\u003e wild-type and genetically modified strains were cultivated on HA plates overlayed with cellophane for three days in the dark or grown on HA media for seven days under constant light for sporulation. Mycelia from the same plate were collected as a biological replicate. DNA-free total RNAs were used for first-strand cDNA synthesis with oligo (dT) and SuperScript III reverse transcriptase (Invitrogen, Thermo Fischer Scientific) according to the manufacturer\u0026rsquo;s instructions. RT reactions were diluted 10 folds with ddH\u003csub\u003e2\u003c/sub\u003eO prior to performing qRT-PCR using SYBR Green (Invitrogen, Thermo Fischer Scientific). \u003cem\u003eB. cinerea Tubulin\u003c/em\u003e (BCIN_01g08040) was used as reference genes to normalize mRNA. Relative transcripts were calculated by 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e based on the previous method \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e using qPCR cycler (Quantstudio5, Thermo Fisher Scientific). Primer sequences were listed in Table \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eDNA-free total RNAs (1\u0026micro;g) were used for a first-strand cDNA synthesis reaction with specific stem-loop RT primer and reverse transcription was carried out as described previously \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e with minor modifications. The resultant cDNA was directly used for amplification using GoTaq DNA Polymerase (Promega) on a thermo cycler initiated with 95\u0026deg;C for 2 min, then 32 cycles of denaturation at 95\u0026deg;C for 30 sec, annealing at 60\u0026deg;C for 30 sec and extension at 72\u0026deg;C for 20\u0026ndash;180 sec (1 kb/min), followed by 5 min extension at 72\u0026deg;C. PCR products were visualized with 10% non-denaturing PAGE gel. Oligonucleotides were provided in Table \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eBcAGO IP\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e strains expressing 3xHA-BcAGO were grown in liquid HA media overnight. 5 g of fresh mycelia were homogenized by mortar and pestle and suspended in 20 ml extraction buffer according to the previous method (20 mM Tris-HCl pH7.5, 300 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 5 mM DTT, 1 tablet cOmplete\u0026reg; protease inhibitor cocktail (Roche)/50 ml, 5 \u0026micro;l RNase inhibitor (40 U)/50 ml) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The lysate was incubated on a vertical wheel and centrifuged for 40 min at 4\u0026deg;C. Mycelia debris was excluded by spinning down and filtered through Miracloth. EZview\u0026trade; Red Anti-HA Affinity Gel (Merck) was used for IP. Samples were incubated at 4\u0026deg;C for 1 h. Beads were pelleted down in a pre-cooled centrifuge at 200 \u0026times; g and washed five times with Wash buffer (20 mM Tris-HCl pH7.5, 300 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X-100, 5 mM DTT, 1 tablet cOmplete\u0026reg; protease inhibitor cocktail/50 ml, 5 \u0026micro;l RNase inhibitor (40 U)/50 ml). Proteins were separated with 8% SDS-polyacrylamide gel at 80 volts for 30 min and 140 volts for 2 h and transferred to PVDF membrane (Immobilon-FL) overnight at 4\u0026deg;C. Transferred membranes were blocked with 10 ml of 5% (v/v) skim fat milk in 1\u0026times; PBS at 4\u0026deg;C for 1 h on a rolling shaker. Membranes were then incubated overnight with primary \u0026alpha;-HA antibody (3F10, Roche). The membranes were incubated with secondary antibody \u0026alpha;-rat IRdye800 (LI-COR) 1 h. Protein signals were detected under Odyssey imaging system (LI-COR).\u003c/p\u003e\n \u003cp\u003eSmall RNA-seq\u003c/p\u003e\n \u003cp\u003eSmall RNA libraries were cloned following manufacturer\u0026rsquo;s instructions (NEBNext Multiplex Small RNA Library Prep for Illumina) and sequenced on an Illumina HiSeq1500 platform. From 20 microgram total RNA extractions, small RNAs were size selected on a 15% polyacrylamide gel electrophoresis, as described previously \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. From BcAGO IP samples, RNA fractions were directly used for small RNA library cloning. Sequencing raw reads were demultiplexed, adapter-trimmed and quality-filtered (q\u0026thinsp;=\u0026thinsp;20). Small RNA reads in the size range of 18\u0026ndash;30 nt were considered for further analysis. Reads were mapped to the reference genome assemblies of \u003cem\u003eB. cinerea\u003c/em\u003e strain B05.10 (Ensembl, ASM83294v1) or tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e accession Heintz SolGenomics Network, version SL4.0). This was performed using ShortStack3 \u003csup\u003e35\u003c/sup\u003e with unique weighting, which uses BOWTIE with zero mismatch (-v 1) as its alignment engine. Reference sequences of rRNA, tRNA, sn/snoRNA, mRNA, repeat RNA for tomato were downloaded from the SolGenomics Network FTP site, and for \u003cem\u003eB. cinerea\u003c/em\u003e from the Ensembl FTP site. \u003cem\u003eB. cinerea\u003c/em\u003e retrotransposon RNA reference sequences were used, as previously annotated \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Small RNA read mapping against different RNA reference sequences was performed using BOWTIE2 \u003csup\u003e57\u003c/sup\u003e. Raw read numbers were normalized to reads per million (RPM) to total read numbers mapped to the respective reference genome. Small RNA cross-kingdom target prediction was conducted with the TAPIR tool \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e using free energy ratio cut-off 0.7 and score cut-off 5.5. Furthermore, no gap, no three mismatches in a row and no two mismatches in a row in the seed region (2\u0026ndash;12 nt) was permitted in the target alignment. Prediction of endogenous mRNA alignments for BcsRNAs was performed using GSTAr (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/MikeAxtell/GSTAr\u003c/span\u003e\u003c/span\u003e), which is based on RNAplex \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. These were filtered to include only targets which are 1) from an BcsRNA that is highly expressed (\u0026gt;\u0026thinsp;40 locus RPM), 2) to an mRNA which is induced in a specific \u003cem\u003ebcago\u003c/em\u003e mutant (p-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.1, L2FC\u0026thinsp;\u0026gt;\u0026thinsp;0.58), and 3) from a BcsRNA that is either reduced in a specific \u003cem\u003ebcago\u003c/em\u003e mutant (p-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.1, L2FC \u0026lt; -0.58) or is frequently bound to an BcAGO via IP (\u0026gt;\u0026thinsp;30 RPM).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eBcsRNA loci annotation\u003c/h2\u003e\n \u003cp\u003eSmall-RNA-loci were defined using a custom pipeline based on genome-wide assessment of depth, outlined in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. Alignments for wildtype libraries (n\u0026thinsp;=\u0026thinsp;2) were used to generate a coverage profile, normalized to RPM. A Poisson distribution was fitted to assess probability that a genomic position has expression higher than background. For this, lambda was calculated for each chromosome separately, based on 40 nucleotide windows with total depth calculated from intergenic regions, using the following formula:\u003c/p\u003e\n \u003cp\u003elambda\u0026thinsp;=\u0026thinsp;window_read_count / intergenic_length * intergenic_read_count\u003c/p\u003e\n \u003cp\u003eIntergenic regions are defined as positions not as featuretype\u0026thinsp;=\u0026thinsp;mRNA in the NCBI gene annotation. Regions are then defined as positions with a Poisson probability of 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e or less and merged if they are \u0026lt;\u0026thinsp;150 nucleotides apart. Merged regions are trimmed to exclude edges which are \u0026lt;\u0026thinsp;5% of their maximum depth. Finally, regions are padded by 10 nucleotides on either edge, resulting in \u003cem\u003eBcSRNA\u003c/em\u003e loci.\u003c/p\u003e\n \u003cp\u003eLoci are assessed in terms of their basic dimensions (length, distance to prior loci) and BcsRNA profile (abundance, RPM, most common BcsRNA sequence and depth, strand preference, and complexity). Size specificity is also assessed, showing the abundance of the most common consecutive sizes for the locus. This is summarized in the field \u0026ldquo;sizecall\u0026rdquo;, which is the smallest number of consecutive sizes that are \u0026gt;\u0026thinsp;50% of the locus abundance, with \u0026ldquo;N\u0026rdquo; indicating loci that are not specific and likely derived from degradation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003emRNA-seq\u003c/h2\u003e\n \u003cp\u003eExtracted total RNA was used for mRNA library cloning with prime-seq method as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. mRNA libraries were sequenced by paired end sequencing on an Illumina HiSeq1500 platform. Raw data was demultiplexed into fastq files using the deML \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e and processed using the zUMIs pipeline (2.9.6) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e with STAR (2.6) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Transcript counts were calculated based on pseudo-alignments using the tool salmon \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and the following command: salmon quant -l SF, with indices based on the ASM83294v1 transcriptome. Raw read counts were transformed using the DESeq2 Bioconductor package \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The regularized logarithm transformation (rlog) was used to transformed reads prior to visualization. Euclidean distance was used for DistHeatMap to calculate the distance within samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e AGO proteins were identified by searching the protein databases for homologies of QDE2 and SMS2 in \u003cem\u003eNeurospora crassa\u003c/em\u003e using BLAST search \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The rooted phylogenetic tree was constructed with amino acid sequences of representative filamentous fungal AGO proteins in phyla of Ascomycota by RAXML method with JTT model. Fungal species and their accession number were listed in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. The alignment was performed by MAFFT and bootstrap was calculated based on 1000 replicates. The phylogenetic tree was built at CIPRES Science Gateway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eData plotting and statistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 10 software was used for plotting and statistical analysis. One-way ANOVA with Tukey multiple comparisons test (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was performed for multi-samples comparison. For two samples comparison, unpaired t-test was performed. Statistical analysis was set for two-tailed p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eData accessibility\u003c/p\u003e\n\u003cp\u003eSequencing data have been deposited in NCBI SRA (BioProject ID PRJNA1092616).\u003c/p\u003e\n\u003cp\u003eAuthors` contribution\u003c/p\u003e\n\u003cp\u003eAW conceptualized this work, raised funding acquisition, led project administration, supervised this work, and wrote the original draft. APC and LH performed investigation, formal analysis, data curation, wrote the original draft and developed methodologies. LO performed formal analysis and developed methodologies. AG and KS participated in investigation, formal analysis. NRJ, FS, LW and WE contributed to formal analysis and data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would like to thank Michael Feldbr\u0026uuml;gge and Claude Becker for critical proofreading of this work. We want to thank the Gene Center Munich for Illumina NextSeq sequencing service. We also would like to thank Martin Parniske for scientific discussions and providing access to the Golden Gate cloning system, Silke Robatzek and Eliana Mor for access to and technical assistance with the DMi8 Thunder Imager microscope. We thank Verena Klingl and Adriana H\u0026ouml;rmann for technical support. AW is supported by the German Research Foundation (DFG), project ID 433194101 in the frame of the research unit RU5116. NRJ is supported by ANID-fondecyt (Chile) #11220727. LH was supported by the China Scholarship Council (CSC).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMeister G. 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Basic local alignment search tool. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 403-410 (1990).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4183067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4183067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArgonaute (AGO) proteins bind to small RNAs to induce RNA interference (RNAi), a conserved gene regulatory mechanism in animal, plant, and fungal kingdoms. Small RNAs of the fungal plant pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e were previously shown to translocate into plant cells and bound to the host AGO, which induced cross-kingdom RNAi to promote infection. However, the role of pathogen AGOs during host infection stayed elusive. In this study, we revealed that members of fungal plant pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e BcAGO family contribute to plant infection and act in bidirectional cross-kingdom RNAi, from fungus to plant and \u003cem\u003evice versa\u003c/em\u003e. Providing these new mechanistic insights of pathogen AGOs promise to improve RNAi-based crop protection strategies.\u003c/p\u003e","manuscriptTitle":"Fungal Argonaute proteins act in bidirectional cross-kingdom RNA interference during plant infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 16:38:09","doi":"10.21203/rs.3.rs-4183067/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2355931-df3b-43e1-848d-2302443239c1","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30031366,"name":"Biological sciences/Molecular biology/RNAi"},{"id":30031367,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2024-09-03T03:55:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 16:38:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4183067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4183067","identity":"rs-4183067","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}