bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Bioinformatic analysis deciphers the molecular toolbox in the endophytic/pathogenic 2 behavior in F. oxysporum f. sp. vanillae - V. planifolia Jacks interaction. 3 Solano De la Cruz MT1**, Escobar – Hernández EE2**, Arciniega – González JA3, Rueda 4 – Zozaya RP1, Adame – García J4, Luna – Rodríguez M5

5 ** These authors contributed equally to this work. 6 1Instituto de Ecología, Universidad Nacional Autónoma de México, Circuito Exterior S/N 7 anexo, Jardín Botánico exterior, Ciudad Universitaria, Ciudad de México, México.

8 2Unidad de Genómica Avanzada, Langebio, Cinvestav, Km 9.6 Libramiento Norte 9 Carretera León 36821, Irapuato, Guanajuato, México.

10 3Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México, 11 Ciudad de México, México.

12 4Tecnológico Nacional de México, Instituto Tecnológico de Úrsulo Galván, Úrsulo Galván, 13 Veracruz, México.

14 5Laboratorio de Genética e Interacciones Planta Microorganismos, Facultad de Ciencias 15 Agrícolas, Universidad Veracruzana. Circuito Gonzalo Aguirre Beltrán s/n, Zona 16 Universitaria, Xalapa, Veracruz, México.

17 Abstract

18 Background:

19 F. oxysporum as a species complex (FOSC) possesses the capacity to specialize into host- 20 specific pathogens known as formae speciales. This with the help of horizontal gene 21 transfer (HGT) between pathogenic and endophytic individuals of FOSC. From these 22 pathogenic forma specialis, F. oxysporum f. sp. vanillae (Fov) is the causal agent of 23 fusarium wilt producing root and stem rot (RSR) and is positioned as the main 24 phytosanitary problem in vanilla plantations worldwide. Nonetheless, the origin of this 25 forma speciales and the behavioral genetics dictating the endophytic/pathogenic Fusarium 26 lifestyles still unknown. To elucidate the underlying molecular mechanisms that establish 27 these behaviors we analyzed the RNA-seq libraries of two-times frames of vanilla-Fov 28 interactions.

29 Results:

30 Our analyses identified the sets of transcripts corresponding to Fov pathogenic strain 31 JAGH3 during the two-times frames of the infection as the sets of the transcripts belonging 32 to endophytic Fox in vanilla. Functional predictions of de novo annotated transcripts as the 33 enriched GO terms with the overrepresented metabolic pathways, allowed us to identify the 34 processes that establish the pathogenic lifestyle in Fov being virulence, hypervirulence, 35 sporulation, conidiation, necrosis and fusaric acid related genes with the carbohydrates, 36 amino acids, proteins, glycerophospholipids and autophagy metabolic pathways that are 37 key regulators of spores germination and pathogenicity establishment as the underlying bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

38 mechanisms behind this behavior. As the absence of these were found in the vanilla 39 endophytic Fox.

40 Conclusions:

41 This work reveals the main players of the behavioral genetics in pathogenic Fov/endophytic 42 Fox in V. planifolia Jacks. Its pathogenic strategy allows Fov to infect in a SIX genes- 43 independent manner. As the other pathogenic elements found in this study could be 44 explained by the presence of pathogenicity islands and genomic regions associated with 45 supernumerary chromosomes in Fov. These play a central role as carriers of genes involved 46 with pathogenic activity and could be obtained through HGT.

47 Key words: formae speciales, horizontal gene transfer, behavior, lifestyles, 48 endophytic/pathogenic. 49 Background

50 The main phytosanitary problem affecting vanilla production worldwide is RSR caused by 51 the phytopathogenic fungus Fov (Pinaria et al., 2015; Tombe et al., 1994, Thomas et al., 52 2002). This fungus has been reported in vanilla growing regions around the world; 53 however, its origin and evolution still poorly understood (Pinaria et al., 2015). Pinaria et al., 54 (2010) reported that the management of the diseases caused by Fox (F. oxysporum) 55 eventually leads to obtaining resistance (Fravel et al., 2003), however in the vanilla crop, 56 the plant material continues to be highly susceptible. The isolates, genetically and 57 morphologically different from Fox, have been classified as special forms depending on the 58 host from which they were obtained, these special forms establish FOSC (Booth, 1971; 59 Michielse and Rep, 2009). Within FOSC exist a broad genomic plasticity, diversifying into 60 different special forms with distinct genomic composition, also having the presence of 61 supernumerary chromosomes among the special forms of FOSC, in fact it has been reported 62 that the presence of certain supernumerary chromosomes, like chromosome 14, bestow 63 pathogenicity to non-pathogenic strains (Ma et al., 2010). Previously it was stated that 64 within each special form, the same genomic structure and the same evolutionary origin 65 were shared (Taylor et al., 1999b) allowing an adequate taxonomic classification based on 66 the sequence of some specific genes (Pinaria et al., 2015; O'Donnell et al., 1998; Taylor et 67 al., 1999a); however, the F. oxysporum f. sp. cubense and F. oxysporum f. sp. vanillae have 68 multiple independent origins (O'Donnell et al., 1998; Flores-de la Rosa et al. 2018) 69 showing the genomic plasticity within FOSC. In fact, Pinaria et al., (2015), mentioned that 70 most of the special forms do not have a monophyletic origin, which suggests significant 71 differences in their genomic structure. Also, the genetic diversity of Fov has been 72 demonstrated by presenting multiple vegetative compatibility groups (Tombe et al., 1994; 73 Pinaria, 2010), as well as several different fingerprint haplotypes (Pinaria et al., 2015). 74 Based on these vegetative compatibility groups (Elias et al., 1993), it was proposed that the 75 evolutionary origin of Fov is complex and not monophyletic having Mexico as the possible 76 center of origin. Fov has been isolated as an endophyte from vanilla stems without any 77 external or internal symptoms and it also shown to be pathogenic in greenhouse trials (Liew 78 et al., 2008).

79 In 2019, our research group reported the increased expression of gene transcripts associated 80 with ribosomal proteins during the early stages of infection caused by Fov in V. planifolia bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

81 Jacks root (Solano De la Cruz et al., 2019). In this work, we found that the RNA libraries in 82 the transcriptome under study contain readings corresponding to Fov, these were aligned 83 against the reference genome of Fox; Fol 4287 (Fusarium oxysporum f. sp. lycopersici). 84 Given those corresponding to Fov, in each of the libraries analyzed being in a range of 85 around 5 to 39%, with the content of the lowest readings in the controls with Fox readings 86 compared to the treatments. Here, we detailed the results of the alignment of Fov JAGH3 87 strain readings to the reference genome Fol 4287, its annotation and the identification of 88 Fov transcripts, present during the Fov infection process in vanilla, at two-time frames 2 89 days post-inoculation (dpi) and 10 dpi. This being the first report of coding sequences 90 corresponding to the proteins with vital functions in the fusarium wilt caused by Fov in 91 vanilla on a large scale. The foregoing as an effort in determining the transcripts present in 92 the JAGH3 strain, which are not present in Fox lineages with endophytic lifestyle in the 93 vanilla root, which could explain its pathogenicity indirectly, as a first approach to 94 obtaining the Fov transcriptomic and genomic data.

95 Results

96 The quality analysis of the libraries corresponding to the V. planifolia Jacks transcriptome 97 in response to the infection caused by Fov, reported by our working group in 2019 (Solano 98 De la Cruz et al., 2019), was again accomplished. Later, the alignment of the libraries 99 against the Fol 4287 genome was performed, obtaining only the readings that correctly 100 aligned against it. About the control libraries, in each of the replicas at 2 dpi 7158305, 101 7249959, and 5741789 aligned readings were acquired. While at 10 dpi 6458564, 6730883, 102 and 6528340 readings were obtained. In 2 dpi, a total of 10917618 aligned readings were 103 collected and at 10 dpi 9397911, 10190151, and 10190151, aligned readings were retrieved. 104 Next, we filtered the alignments results by alignment quality and sorted them by genomic 105 coordinates.

106 To be able to identify the genes of the 2 dpi and 10 dpi controls and treatments, we carried 107 out intersections of the filtered readings with the annotation of Fol 4287 genome utilizing 108 the genomic coordinates. Table 1 presents the number of genes identified corresponding to 109 each one of them. Finally, we determined the annotated genes that are shared between 110 treatments and the treatment-specific ones. From the annotated transcripts we used their 111 identifiers to retrieve their protein sequences from Biomart-Ensembl Fungi database for 112 each of the libraries. For each of the obtained coding protein data sets. After that, a de novo 113 functional annotation was performed using InterproScan 5 software (Jones et al., 2014).

114 Here, we were able to determine a collection of specific Fov transcripts, which were found 115 only in the vanilla roots that were inoculated with the JAGH3 strain being directly related 116 to the pathogenicity of Fov in V. planifolia Jacks (see in supplementary material the files 117 10_dpi_treatment_genes_list.txt and 2_dpi_treatment_genes_list.txt).

118 Aligned genomic regions revealed the participation of supernumerary chromosomes 119 in the establishment of the pathogenicity of Fov in V. planifolia Jacks

120 In figure 1 we observed the Fox transcripts corresponding to the control treatment, and their 121 location on the different chromosomes in the Fol 4287 reference genome. Interestingly, we 122 observed that many of the transcripts corresponding to endophytic Fox were mainly located bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

123 in the chromosomes that establish the core of the reference Fol 4287 genome and not in the 124 supernumerary chromosomes where we observed the presence of few transcripts. In the 125 control treatment at 2 dpi, we observed the presence of transcripts corresponding mainly to 126 chromosomes 1, 2, 4, 5, 7, 8, 9 and 10 with the presence of some transcripts in 127 chromosomes 3, 6, 11, 12, 13 and 14; while they did not present any transcript related to 128 chromosome 15. In the control treatment at 10 dpi, the most represented chromosomes are 129 1, 2, 4, 5, 7, 8, 9 and 10, while some transcripts related to chromosome 3, 11, 12 and 13 130 were found; Interestingly, transcripts related to chromosomes 6, 14, and 15 were absent.

131 In figure 2 we detected the transcripts corresponding to the libraries of the root treatment 132 that were inoculated with the JAGH3 strain at 2 dpi and their location in the Fol 4287 133 reference genome.

134 Concerning the treatments where the JAGH3 strain was inoculated, in the Fol 4287 135 karyotype we found a greater number of transcripts present in each of the chromosomes, 136 not only in the core genome but also in the supernumerary chromosomes. In fact, in the 137 treatment at 2 dpi, in the core genome, we found the presence of transcripts in 138 chromosomes 1, 2, 4, 5, 7, 8 and 9 with a clear increase in the number of transcripts present 139 in each of these chromosomes against the control, being even more contrasting the increase 140 in the number of transcripts observed in chromosomes 10, 11, 12, 13 and 14. The latter 141 being considered as supernumeraries and associated with pathogenicity in Fox.

142 Finally, this increase in the number of transcripts present in the Fol 4287 karyotype 143 observed in the treatment with the JAGH3 strain was evidenced in a contrasting way 144 compared to the control treatment, at 10 dpi, where we found a significant increase on the 145 core genome chromosomes and in the supernumerary chromosomes.

146 A conspicuous aspect that we must remark is the presence of components of pathogenicity 147 islands in Supplementary Figure 1, present only in the libraries of the treatment with the 148 JAGH3 strain at 10 dpi. Thus, showing the participation of this component in late infection 149 and could be associated with the establishment of the necrotrophic behavior during the 150 infection caused by Fov in vanilla.

151 Metabolic pathways of the JAGH3 strain related to the infection process of the V. 152 planifolia Jacks root

153 To elucidate the metabolic pathways in which the annotated Fov JAGH3 strain genes may 154 be playing a crucial role during the infection in the vanilla root. We performed a functional 155 prediction annotation and metabolic mapping using KEGG database (Kanehisa and Goto, 156 2000) (see supplementary material). Thus, the number of genes identified in the present 157 work having a role in each of the pathways found was also obtained. In the supplementary 158 table 1 and the supplementary table 2 there is a list of the metabolic pathways, and the 159 number of genes associated with each pathway during early infection at 2 dpi (see 160 Metabolic_routes_of_early_infection_at_2_dpi.xlsx) as well as late infection 10 dpi (see 161 All_the_metabolic_routes_of_the_infection_dead_10_days.xlsx), respectively.

162 Interestingly, the number of genes corresponding to each of the metabolic pathways 163 described above was considerably higher during late infection compared to early infection. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

164 On the other hand, at 10 dpi we determined the presence of genes corresponding to the 165 following metabolic pathways, which are only found during late infection: Penicillin and 166 cephalosporin biosynthesis; Homologous recombination; Biotin metabolism; D-Arginine 167 and D-ornithine metabolism; Atrazine degradation; Synthesis and degradation of ketone 168 bodies; Sulfur relay system; C5-Branched dibasic acid metabolism; Polyketide sugar unit 169 biosynthesis; Carotenoid biosynthesis; Selenocompound metabolism; Vitamin B6 170 metabolism; Carbapenem biosynthesis; Sesquiterpenoid and triterpenoid biosynthesis.

171 Gene Ontology and KEGG Gene Enrichment analysis offer crucial insights about Fov 172 pathogenesis in V. planifolia Jacks

173 Concerning the biological processes of GO analysis, we obtained 125 and 43 statistically 174 significant at 2 dpi and 10 dpi, respectively. Where at 2 dpi (Figure 3) we observed terms 175 involved in sporulation such as cell wall biogenesis and external encapsulating structure 176 organization terms. Also, mechanisms possibly linked to virulence and infection as we 177 found terms associated with protein, peptide, and organic substance transport. At 10 dpi 178 (Figure 4) there were mostly regulatory mechanisms of nucleic acids, metabolic processes 179 of macromolecules and organic substances and terms possibly related to sporulation 180 including cell cycle. These data suggest that pathogenic mechanisms like sporulation, 181 toxins and pathogenic effectors remain active along the infection process from early to late 182 stages.

183 Regarding the metabolism, we found 21 and 46 statistically significant metabolic pathways 184 at 2 dpi and 10 dpi, respectively. At 2 dpi (Figure 5) we observed pathways involved with 185 reproduction and biosynthesis of metabolites and other biomolecules. In a similar manner 186 we found pathways linked with these processes at 10 dpi (Figure 6). Additionally, there 187 were other types of biomolecules synthesized at the late stage of infection. These results 188 further confirm the previous found in GO analysis where biological processes and now 189 metabolic pathways are associated with pathogenesis and stay operating along the infection, 190 thus explaining the pathogenic behavior of Fov.

191 De novo annotation as a functional prediction of Fov transcripts shed light on the role 192 of pathogenesis related genes in the plant-host interaction.

193 To characterize the molecular players that may play a main role during the pathogenesis in 194 vanilla we did a de novo annotation as a functional prediction of Fov genes during these 195 processes, firstly categorizing the shared and not shared genes between these two-time 196 frames (Table 2).

197 Among the annotated genes, we found virulence and hypervirulence factors during both 198 stages of infection, interestingly these virulence factors increase in number and types 199 during the late stage of infection (Table 3) in contrast with the early stage of infection 200 (Table 4), this could mean that different virulence factors participate during different time 201 frames along the disease.

202 Additionally, at 2 dpi we obtained genes associated with sporulation (Table 5), 203 necrotic/pathogenic activity (Table 6) and fusaric acid (Table 7). These results further 204 confirm the molecular mechanisms which establish the pathogenic toolbox used by Fov 205 during early infection stages. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

206 Meanwhile, at 10 dpi genes involved in pathogenic processes found at 2 dpi were observed. 207 However, there were remarkable differences in comparison with the late stage of infection, 208 where there was conidiation (Table 8), pathogen-effector (Table 9) and Fusarinine (Table 209 10) related genes that were absent during the early stage of infection. Therefore, suggesting 210 that these pathogenic processes are late stage exclusive in Fov-V. planifolia Jacks 211 pathosystem.

212 Discussion

213 Use of sugar as an energy source and the growth of conidia in early Fox infection in 214 vanilla root.

215 As previously described, the synthesis of carbohydrates, as well as the production of sugar, 216 alcohol and organic acids is one of the most active metabolic pathways of Fox metabolism 217 during the germination process of conidia (Sharma et al., 2016). This, being one of the first 218 differences that we observed in this work in relation to the control treatment, where the 219 pathogenic strain JAGH3 was not inoculated. The significant presence of genes 220 corresponding to the synthesis of carbohydrates, production of alcohol and organic acids in 221 the treatments with the JAGH3 strain in both time frames of infection was detected. We 222 also showed the presence of transcripts related to carbohydrate metabolism, oxidative 223 phosphorylation, glycolysis, and the pentose phosphate pathway as metabolic pathways 224 related to the germination of Fox conidia (Sharma et al., 2016). In addition to the 225 mentioned study, there is evidence that sugar, as an easily oxidized carbon source, supplies 226 the demand for the conidia germination process (Cochrane et al., 1963; Griffin 1970a; and 227 Griffin, 1970b). In fact, at 2 dpi we found the presence of genes of hexokinase, trehalose 6- 228 phosphate synthase, beta-fructofuranosidase, neutral trehalase, murein transglycosylase, 229 alpha-trehalose-phosphate synthase, trehalose 6-phosphate synthase, endoglucanase type B, 230 beta-glucosidase, glucoamylase, alpha-amylase, glycosyl hydrolase family, corresponding 231 to the metabolism of starch and sucrose (see supplementary material). The above is 232 evidence of the use of an energy source during Fov infection in vanilla root, which previous 233 works have associated with the growth of hyphae in Fox during the germination of conidia, 234 a biological process that is associated with the early stages of infection.

235 At 10 dpi we found the following genes linked to the metabolism of starch and sucrose: 236 beta-glucosidase, murein transglycosylase, glucan 1,6-alpha-glucosidase, beta-glucosidase 237 G, trehalase, beta-glucosidase, alpha-glucosidase, endoglucanase, beta-glucosidase K, 238 glycogen phosphorylase, endoglucanase, oligo-1,6-glucosidase, alpha alpha-trehalse, 239 glucan 1,3-beta-glucosidase. In addition to the genes involved with glycolysis and 240 gluconeogenesis: alcohol dehydrogenase (NADP +), aldehyde dehydrogenase (NAD +), 241 pyruvate decarboxylase, aldose 1-epimerase, which prefer propanol. This shows the 242 involvement of carbohydrate metabolism in the late phase of Fov infection in vanilla.

243 Amino acid metabolism in Fox infection in vanilla root

244 The participation of amino acid metabolism in plant-pathogen interaction and its 245 participation in the establishment of pathogenicity has been previously documented 246 (Phillips et al., 2004; Jonkers et al., 2009; Scandiani et al., 2014; Tchameni et al., 2012; 247 Okada and Matsubara, 2021; Kasote et al., 2020). At 2 dpi, different genes related to amino bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

248 acid biosynthesis were determined, such as: aspartate kinase; pyruvate carboxylase; 249 tagatose 1,6-diphosphate aldolase; asparagine synthetase; ribose-phosphate 250 pyrophosphokinase 3, 6-phosphofructokinase, acetylornithine aminotransferase, 251 mitochondrial; tryptophan synthase and branched-chain amino acid aminotransferase. 252 Jastrzębowska and Gabriel in 2015, proved that the inhibition of amino acid biosynthesis, 253 by means of chemical substances that act as antifungals (N- (5-substituted-1,3,4-thiadiazol- 254 2-yl) cyclo- propanecarboxamides), significantly reduces Fox growth in minimal medium. 255 Likewise, these authors refer that auxotrophic mutants of human pathogenic fungi impaired 256 in biosynthesis of amino acids exhibit growth defects or at least reduced virulence under in 257 vivo conditions (Jastrzębowska and Gabriel, 2015). At 10 dpi, we also confirmed the 258 participation of genes associated to amino acid biosynthesis in the growth of conidia, by 259 determining the presence of genes corresponding to: cystathionine gamma-lyase; 260 anthranilate phosphoribosyltransferase; cysteine synthase A; mitochondrial homocitrate 261 synthase; argininosuccinate lyase; dTDP-4-dehydrorhamnose reductase; 3-isopropylmalate 262 dehydrogenase; dihydroxy-acid dehydratase, mitochondrial; ribose 5-phosphate isomerase 263 B; serine hydroxymethyltransferase, mitochondrial; homoserine dehydrogenase; threonine 264 aldolase; imidazoleglycerol-phosphate dehydratase; argininosuccinate synthase; 265 acetylornithine deacetylase; dihydrodipicolinate synthase; glutamine synthetase; 266 homoisocitrate dehydrogenase; pyrroline-5-carboxylate reductase; tryptophan synthase, 267 beta subunit; 3-dehydroquinate synthase; L-2-aminoadipate reductase; glycine 268 hydroxymethyltransferase; S-adenosylmethionine synthetase; 3-deoxy-7- 269 phosphoheptulonate synthase; threonine dehydratase.

270 Also, at 2 dpi we found the presence of genes linked to the synthesis of tyrosine such as: 271 acylpyruvate hydrolase, primary-amine oxidase, aldehyde dehydrogenase (NAD +), 272 tyrosinase, gentisate 1,2-dioxygenase, 4-hydroxyphenylpyruvate dioxygenase, primary - 273 amine oxidase, homogentisate 1,2-dioxygenase, and fumarylacetoacetase. Whose presence 274 and importance in the genomic context were reported in filamentous fungi Greene et al., 275 (2014). For its part, at 10 dpi, we detected the following genes involved with the tyrosine 276 metabolism pathway: catechol O-methyltransferase, succinate-semialdehyde 277 dehydrogenase (NADP +); maleylacetoacetate isomerase; copper amine oxidase; succinate- 278 semialdehyde dehydrogenase / glutarate-semialdehyde dehydrogenase; monoamine 279 oxidase; 1,2-dioxygenase homogentisate; alcohol dehydrogenase, propanol-preferring; and 280 aspartate aminotransferase, cytoplasmic.

281 At 10 dpi we observed transcripts related to arginine and proline metabolism: glutamate 5- 282 kinase; 1-pyrroline-5-carboxylate dehydrogenase; ornithine aminotransferase; 1-pyrroline- 283 5-carboxylate dehydrogenase; N1-acetylpolyamine oxidase; N1-acetylpolyamine oxidase; 284 aldehyde dehydrogenase (NAD +); D-amino-acid oxidase; monoamine oxidase; proC; 1- 285 pyrroline-5-carboxylate dehydrogenase; amidase; ornithine decarboxylase; agmatine 286 deiminase; polyamine oxidase; aspartate aminotransferase, cytoplasmic; 4-hydroxy-2- 287 oxoglutarate aldolase. Boedi et al in 2016 reported the comparison by transcriptome 288 analysis of the pathogenic (biotrophic) phase against the saprophytic phase in F. 289 graminearum (Boedi et al., 2016). Finding in pathogenesis an increase in the order of at 290 least two times in magnitude in the expression of genes corresponding to the arginine and 291 proline metabolism pathway. Indicating that the metabolism of arginine and proline plays bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

292 an important role in the growth of hyphae, participating in the biotrophic phase of Fox in 293 plants.

294 Protein metabolism in Fox infection in vanilla root

295 Concerning the protein metabolism its participation in the establishment of pathogenicity 296 has been widely reported (Kumar et al., 2016; Kong et al., 2019). We found at 2 dpi the 297 presence of genes associated with the processing of proteins in the endoplasmic reticulum, 298 such as: nuclear protein localization protein 4, oligosaccharyl transferase complex subunit 299 OST4, protein disulfide-isomerase erp38, translocation protein SEC63, protein transporter 300 SEC23, mannosyl-oligosaccharide alpha-1,2-mannosidase, hypothetical protein, 301 hypothetical protein, protein disulfide-isomerase, Cullin 1, hypothetical protein, 302 hypothetical protein, translation initiation factor 2 subunit 1, IRE protein kinase, DnaJ like 303 subfamily B member 12, protein transporter SEC61 subunit beta, protein OS-9. Wang et 304 al., (2020) report that the use of myriocin as an antifungal negatively affects the stability of 305 the membrane of F. oxysporum f. sp. niveum. When analyzing the combined analysis 306 between the transcriptome and proteome revealed that the expression of some membrane- 307 related genes and proteins, mainly those associated with sphingolipid metabolism, 308 glycerophospholipid metabolism, steroid biosynthesis, ABC transporters and protein 309 processing in the endoplasmic reticulum, was disordered.

310 Also, in the treatment of 2 dpi we detected the presence of genes linked to proteolysis 311 mediated by ubiquitination, these genes correspond to: F-box and WD-40 domain- 312 containing protein MET30, F-box and WD-40 domain-containing protein CDC4, ubiquitin- 313 conjugating enzyme (huntingtin interacting protein 2), SUMO-conjugating enzyme ubc9, 314 Cullin 1, ubiquitin-like 1-activating enzyme E1 B, ubiquitin-conjugating enzyme E2 R and 315 pre-mRNA-processing factor 19. Recently, numerous studies revealed that F-box proteins 316 are required for fungal pathogenicity (Liu and Xue, 2011). Proteome analysis in F. 317 oxysporum showed the role of Fb1 protein in proteasome degradation and susceptibility as 318 potential targets of ubiquitination (Manikandan et al., 2018; Miguel-Rojas and Hera, 2013). 319 A proteomic approach in F. oxysporum f.sp. lycopersici was done by Miguel-Rojas and 320 Hera (2013) where they identified the differential pattern of proteins involved in vesicle 321 blocking and proteasome degradation (Manikandan et al., 2018). The function of F-box 322 proteins and their potential as part of the ubiquitin-proteasome system (UPS) has been 323 reported in several fungal pathogens, including those that cause human infections (such as 324 C. neoformans and C. albicans) or plant diseases (such as Fusarium species and M. oryzae) 325 (Liu and Xue, 2011). Fungi such as Fusarium species contain much larger numbers of F- 326 box proteins than yeast, approximately 60~94 in Fusarium species compared to around 20 327 in yeasts (S. cerevisiae, C. albicans, or C. neoformans). Suggesting that F-box proteins 328 contribute to the regulation of a more complex developmental and metabolic processes 329 occurring in filamentous fungi (Liu and Xue, 2011).

330 Autophagy in Fox infection in vanilla root

331 Autophagy has been described as one of the main routes of cell traffic and recycling, also 332 playing a relevant role in pathogenesis (Lv et al., 2017). These authors, in 2017 reported 333 genome-wide identification and characterization of autophagy-related genes (ATGs) in the 334 wheat pathogenic fungus F. graminearum identifying 28 genes associated with the bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

335 regulation and operation of autophagy; also finding that subsets of autophagy genes were 336 necessary for asexual / sexual differentiation and deoxynivalenol (DON) production, 337 respectively. Finally, they concluded that autophagy plays a critical function in growth, 338 asexual / sexual sporulation, deoxynivalenol production and virulence in F. graminearum. 339 At 2 dpi we found the presence of genes involved with the autophagy pathway such as: 340 CAMK / CAMKL / AMPK protein kinase, hypothetical protein, synaptobrevin, serine / 341 threonine-protein phosphatase PP2A catalytic subunit, Ras-like protein, translation 342 initiation factor 2 subunit 1, CAMKK protein kinase. Kalhid et al., in 2019, found that 343 autophagy assumes an imperative job in affecting the morphology, development, 344 improvement and pathogenicity of Fox, through the study of an ATG gene that regulates its 345 pathogenicity. Corral-Ramos et al., in 2015 demonstrated that autophagy mediates nuclear 346 degradation after hyphal fusion and possesses a general function in the control of nuclear 347 distribution in Fox; in a process regulated by ATG genes; during the vegetative growth of 348 hyphae.

349 Glycerophospholipid in Fox infection in vanilla root

350 In the present work we found the presence at 2 dpi of genes related to the 351 glycerophospholipid metabolism pathway, as widely reported previously (van Aarle and 352 Olsson, 2003; Bhandari et al., 2018; Zhang et al., 2020), such as: ethanolamine kinase, 353 acetyltransferase, phosphatidylserine decarboxylase, phosphatidylserine decarboxylase, 354 lysophospholipase NTE1, CDP-diacylglycerol-inositol 3-phosphatidyltransferase, 355 ethanolamine-phosphate cytidylyltransferase, and NTE family protein. Joshi and Mathur in 356 1987 mentioned that Fox synthesizes up to 26.4% lipids in minimal medium, being 357 glycerophospholipids and triacylglycerols, the latter predominating. The main 358 glycerophospholipid is phosphatidylcholine, the others are phosphatidylethanolamine and 359 phosphatidylinositol. Wang et al, reported in 2020, that Bacillus amyloliquefaciens LZN01 360 Myrozin can inhibit the growth of F. oxysporum f. sp. leveum. In a combined proteome and 361 transcriptome analysis they found that it had expression of some membrane-related genes 362 and proteins, mainly those related to sphingolipid metabolism, glycerophospholipid 363 metabolism, steroid biosynthesis, ABC transporters and protein processing in the 364 endoplasmic reticulum, was disordered. They concluded that myriocin has a significant 365 antifungal activity due to its ability to induce membrane damage in this fungus. Zuo et al, in 366 2017 reported that sterol and glycerophospholipids are important cellular or subcellular 367 membrane components and are required for cell growth and proliferation, maintenance of 368 organelle morphology, signal transduction, and lipid homeostasis (Kihara and Igarashi, 369 2004; Nebauer et al., 2004). Furthermore, said authors reported that exposure to DT from 370 Foc TR4 regulates the metabolism of steroids and glycerophospholipids, affecting the 371 integrity of the cell membrane of FOC TR4 causing DT toxicity (Zuo et al., 2017). In late 372 infection at 10 dpi, we found the following genes associated with the synthesis of 373 Glycerophospholipid acetyltransferase, choline-phosphate cytidylyltransferase, 374 phosphatidylserine decarboxylase, phosphatidylserine synthase, phosphatidylethansolipid 375 synthey N-phosphatidylettransferase 3-methylhalserine-phosphate-synthase-3, 376 phosphatidylethansinehydrogenase-3, phosphatidylethansinehydro-synthase-3 -3-phosphate 377 dehydrogenase, phospholipase A2, triacylglycerol lipase, phosphatidylserine 378 decarboxylase, glycerol-3-phosphate dehydrogenase, ethanolamine kinase, 379 acetylcholinesterase, phospholipase D1 / 2, phosphatidylaglycerine decarboxylasecerine bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

380 A2, phosphatidylaglysecerine decarboxysecerine A2, phosphatidylaglysecerine 381 decarboxysecerine A2, phosphatidylaglysecerine decarboxysecerine 1 -serine O- 382 phosphatidyltransferase, diacylglycerol diphosphate phosphatase / phosphatidate 383 phosphatase.

384 Bioinformatic analysis results throw light on the underlying molecular mechanisms 385 used in pathogenicity in the Fov-V. planifolia Jacks pathosystem.

386 In FOSC, the infection has been described in F. oxysporum f. sp. lycopersici. This 387 pathogenic process is divided into four main events. It starts with the hyphae adhesion to 388 the plant surface, continuing with the penetration of the plant structure which depends on 389 biotic factors and environmental cues, considering that F. oxysporum is an opportunistic 390 pathogen it needs an injury to enter (Bani et al., 2018). Once the fungus has established 391 itself in the plant, the colonization process will begin, there mycelium and spores advance 392 towards xylem vessels causing conidiation to start, later the spores and conidia are transport 393 with the sap via xylem vessels, so these spores germinate and develop into conidiophores 394 and conidia going through the perforation plates. Finally, the pathogen causes the occlusion 395 of xylem vessels and hydric stress by the accumulation of mycelium and toxins, this being 396 reflected in a variety of symptoms including: vascular wilt, abscission, chlorosis, and 397 necrosis. Therefore, the death of the plant. (MacHardy and Beckman, 1981; Bishop and 398 Cooper, 1983; Beckman, 1989; Olivain and Alabouvette, 1997; Lucas, 1998; Olivain and 399 Alabouvette, 1999; Schneider and Carlquist, 2004; Groenewald, 2006; Olivain et al., 400 2006).

401 About the description of the infection in vanilla, there exists few reports of F. oxysporum f. 402 sp. radicis-vanillae affecting this orchid. The symptoms start with the browning and death 403 of underground and aerial roots, continuing with wrinkles in leaves and stem and ending 404 with the death of the plant. As for the infection timing, it requires two days to achieve 405 hyphae adhesion and eight days to complete the colonization phase (Koyyappurath et al., 406 2015; Koyyappurath et al., 2016).

407 In concurrence with the findings in F. oxysporum f. sp. radicis-vanillae, where they 408 documented the germination of conidia and subsequent triggering of the infection at 2 dpi 409 and the colonization of the root later frames of the disease (Koyyappurath et al., 2015). In 410 this study we report, also at 2 dpi the presence of genes related to sporulation, fusaric acid, 411 necrosis, virulence and hypervirulence factors. Thus, explaining the beginning of conidia 412 germination and the genesis of the infection as these other pathogenesis-related genes act as 413 key players during this early plant-host interaction. Also, in agreement, in the advanced 414 stage of infection at 7 dpi and 9 dpi there was progression of the plant colonization and 415 softening of the root tissue as consequence of the fusarium wilt. These symptoms could be 416 justified with the data shown at 10 dpi, here we found genes related to conidiation, 417 pathogenic effectors and fusarinine in addition to those reported at 2 dpi. These are 418 foremostly linked to pathogenesis and tissular death. Therefore, exhibiting a predominant 419 necrotic behavior as reported in this forma specialis (Koyyappurath et al., 2015; 420 Koyyappurath et al., 2016). As these results indicate the possible pathogenic strategy of 421 Fov we summarized it into a schematic representation (Figure 7). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

422 As growing evidence supports the hypothesis that pathogenicity of FOSC relies on 423 transferable extra chromosomes and genes coding for Secreted in Xylem proteins also 424 known as SIX (Ma et al., 2013). It was shown in this study the presence of genes belonging 425 to pathogenicity chromosomes and pathogenicity islands as this hypothesis suggests. 426 Nonetheless, we did not report the presence of SIX genes, therefore despite the similarities 427 in the pathogenic strategy with other formae speciales, the findings in this work suggest 428 that F. oxysporum f. sp. vanillae pathogenic capacity is SIX genes-independent but another 429 pathogenic elements-dependent such as the previously described.

430 Conclusions

431 The bioinformatic analysis of Fov transcripts during the infection in V. planifolia Jacks 432 revealed that this forma specialis make use different pathogenesis associated genes along 433 this process, among them we must remark sporulation, conidiation, fusaric acid, necrosis, 434 pathogenic and virulence effectors as main molecular players. Events that are associated 435 with primary metabolism, carbohydrates, amino acid metabolism, autophagy, and 436 secondary metabolism. Hence, we propose that these allow F. oxysporum f. sp. vanillae to 437 infect vanilla in a SIX genes-independent manner. Also, the presence of them could be 438 explained by the pathogenicity islands and the genomic regions associated with 439 supernumerary chromosomes found in Fov. Both act as carriers of genes involved with 440 pathogenic activity. The results shown here highlight the key players that confer the 441 capacity to infect vanilla to the deadliest pathogen affecting this crop.

442 Methods

443 Experimental framework

444 The experimental framework, that included the plant material collection, infectivity assays, 445 total RNA extraction and the generation with the sequencing of cDNA libraries were done 446 in our previous work in Solano-De la Cruz et al., 2019. The generated data was submitted 447 to the GEO platform of NCBI-GenBank (Accession number: GSE134155).

448 RNA-seq data processing: cleaning, alignment, and filtering

449 Quality of reads obtained from the high-throughput sequences was carried out using 450 FASTQC version 0.11.2 (Andrews, 2010) and MultiQC version 1.0 (Ewels et al., 2016) 451 software. Reads above 32 nt, without the presence of adapters were considered for further 452 analysis. First, to filter and discard reads corresponding to the plant host, genome index and 453 alignment of reads was done utilizing the STAR program version 2.7.2 (Dobin et al., 2012) 454 using the reference genome of F. oxysporum f. sp. lycopersici strain Fol 4287 available on 455 Ensembl Fungi and visualize the alignment output with MultiQC version 1.0 software. To 456 further filter them based on alignment quality, Samtools version 1.9 (Li et al., 2009) and 457 MultiQC for post-filtering visualization, were used.

458 Genes identification and visualization of aligned genomic regions of Fov to Fol 4287 459 karyotype

460 To be able to identify the Fov transcripts participating in the infection, the GenIDs 461 obtention was accomplished. To accomplish this, the intersection of the genomic bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

462 coordinates of the filtered alignment results against the F. oxysporum f. sp. lycopersici 463 strain Fol 4287 genome annotation was done using Bedtools version 2.25.0 (Quinlan and 464 Hall, 2010), with the extraction of GenIDs lists using cut command and AWK, was done. 465 On the other hand, the bed files with the genomic coordinates were used to map and 466 visualize the Fov found genes in the F. oxysporum f. sp. lycopersici strain Fol 4287 467 karyotype using the visualization tool from Ensembl Fungi of Ensembl Genomes 468 consortium (Howe et al., 2020) (available at 469 https://fungi.ensembl.org/Fusarium_oxysporum/Location/Genome).

470 Metabolic description of identified genes: annotation and mapping

471 To elucidate the metabolic pathways where the identified genes could be involved, an 472 annotation and mapping of these biochemical processes were conducted. First, extracting 473 the protein sequences of the identified genes using the Biomart-Ensembl Fungi database 474 (Howe et al., 2020) to annotate them with the BlastKoala tool from KEGG platform 475 (Kanehisa and Goto, 2000) (available at 476 https://www.kegg.jp/kegg/tool/annotate_sequence.html). Later, mapping the annotation 477 against the species-specific KEGG pathway maps belonging to F. oxysporum with the 478 Search Pathway tool from KEGG platform (available at 479 https://www.genome.jp/kegg/tool/map_pathway1.html).

480 Gene Ontology Enrichment Analysis

481 GO enrichment analysis was executed using the topGO R package (Alexa et al., 2006), we 482 applied the following criteria: p-value < 0.05 threshold, algorithm “classic” and “fisher” 483 statistic. We utilized as query the obtained GeneIDs lists from F. oxysporum f. sp. 484 lycopersici strain Fol 4287 in the Biomart-Ensembl Fungi database (Howe et al., 2020). 485 Finally, we visualized this data via RStudio version 4.0.1 and ggplot2 package version 486 3.3.2 (Wickham, 2016).

487 KEGG Gene Enrichment Analysis

488 We did the KEGG enrichment analysis using RStudio and the clusterProfiler package (Yu 489 et al., 2012). To use the enrichKEGG function in this package, we used as input the KO 490 identifiers obtained from BlastKoala annotation and filtered our results by applying 491 adjusted p-value cut-offs < 0.05. For the visualization we used RStudio version 4.0.1 and 492 ggplot2 R package version 3.3.2 (Wickham, 2016).

493 De novo annotation of Fov identified genes.

494 To decipher the potential functions of Fov gene transcripts found from out alignment 495 against the Fol 4287 strain genome. We utilized the software InterproScan 5 version 5.41 496 (Jones et al., 2014). Which allows protein-coding sequences function prediction, using as 497 reference databases Pfam and SUPERFAMILY.

498 Abbreviations bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

499 FOSC: Fox species complex; HGT: horizontal gene transfer; RSR: root and stem rot; Fov: 500 F. oxysporum f. sp. vanillae; Fox: F. oxysporum; Fol: F. oxysporum f. sp. lycopersici; dpi: 501 days post-inoculation; DON: deoxynivalenol.

502 Acknowledgments

503 We are grateful to Dr. Carlos Lobato Tapia and Dr. Rosalinda Tapia López for reviewing 504 this manuscript. To the University Massive Sequencing Unit of the Institute of 505 Biotechnology, UNAM. To M.C Jerome Jean Verleyen for his help in the use of the 506 Teopanzolco bioinformatics cluster. To the National Council of Science and Technology 507 (CONACYT), for the scholarship 720145 granted to the first author EEEH to carry out his 508 postgraduate studies. To the National Laboratory of Genomics for Biodiversity 509 (LANGEBIO), CINVESTAV for the MAZORKA bioinformatic cluster services.

510 Author’s contributions

511 MTSC, EEEH, MLR coordinated the study. MTSC, EEEH and MLR conceived and 512 designed the experiments. MTSC and EEEH performed the bioinformatic analysis. MTSC 513 and EEEH interpreted the results of the analyses. MTSC, EEEH, RPRZ, JAAG, JAG and 514 MLR conceived and organized the manuscript structure. All authors contributed during the 515 manuscript preparation and approved the final manuscript.

516 Funding

517 This work was funded in part by CONACYT (EEEH: CONACYT MSc. Scholarship - 518 720145).

519 Availability of data and materials

520 The datasets generated and/or analyzed during the current study are available in the GEO 521 repository with accession number GSE134155 522 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134155).

523 Ethics approval and consent to participate.

524 Not applicable.

525 Consent for publication

526 Not applicable.

527 Competing interests

528 The authors declare that they have no competing interests.

529 530 References

531 1. Alexa A, Rahnenführer J and Lengauer T. Improved scoring of functional groups 532 from gene expression data by decorrelating GO graph structure. Bioinformatics. 533 2006, 22(13):1600–1607. https://doi.org/10.1093/bioinformatics/btl140. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

534 2. Andrews S. (2010). FastQC: a quality control tool for high throughput sequence 535 data. Available online at: 536 http://www.bioinformatics.babraham.ac.uk/projects/fastqc. 537 3. Beckman C. Colonization of the Vascular System of Plants by Fungal Wilt 538 Pathogens: A Basis for Modeling the Interactions between Host and Parasite in 539 Time and Space. Berlin, Heidelberg: Springer. 1989, p. 19-32. 540 4. Bani M, Pérez-De-Luque A, Rubiales D, Rispail N. Physical, and chemical barriers 541 in root tissues contribute to quantitative resistance to Fusarium oxysporum f. sp. pisi 542 in pea. Front Plant Sci. 2018, 9(2):1–16. https://doi.org/10.3389/fpls.2018.00199. 543 5. Bhandari DR, Wang Q, Li B, et al. Histology-guided high-resolution AP-SMALDI 544 mass spectrometry imaging of wheat-Fusarium graminearum interaction at the root– 545 shoot junction. Plant Methods. 2018; 14, 103. https://doi.org/10.1186/s13007-018- 546 0368-6 547 6. Bishop C and Cooper R. An ultrastructural study of root invasion in three vascular 548 wilt diseases. Physiological Plant Pathology. 1983, 22(1):15-27. 549 https://doi.org/10.1016/S0048-4059(83)81034-0. 550 7. Boedi S, Berger H, Sieber C, Münsterkötter M, Maloku I, Warth B, Sulyok M, 551 Lemmens M, Schuhmacher R, Güldener U and Strauss J. Comparison of Fusarium 552 graminearum Transcriptomes on Living or Dead Wheat Differentiates Substrate- 553 Responsive and Defense-Responsive Genes. Frontiers in Microbiology. 2016, 7: 1- 554 24. https://doi.org/10.3389/fmicb.2016.01113. 555 8. Booth C. The Genus Fusarium. London, UK: Kew UK Commonwealth 556 Mycological Institute. 1971. 557 9. Cochrane V, Cochrane J, Vogel J and Coles R. Spore germination and carbon 558 metabolism in Fusarium solani IV metabolism of ethanol and acetate. Journal of 559 Bacteriology. 1963, 86: 312-319. https://doi.org/10.1128/JB.86.2.312-319.1963. 560 10. Corral-Ramos C, Roca MG, Di Pietro A, Roncero MI, Ruiz-Roldan C. Autophagy 561 contributes to regulation of nuclear dynamics during vegetative growth and hyphal 562 fusion in Fusarium oxysporum. Autophagy. 2015, 11:131-44; PMID:25560310; 563 http://dx.doi.org/10.4161/15548627.2014.994413 564 11. Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson 565 M and Gingeras T. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 566 2013, 29(1): 15-21. https://doi.org/10.1093/bioinformatics/bts635. 567 12. Elias K, Zamir D, Lichtman-Pleban T and Katan T. Population structure of 568 Fusarium oxysporum f.sp. lycopersici: restriction fragment length polymorphisms 569 provide genetic evidence that vegetative compatibility group is an indicator of 570 evolutionary origin. Molecular Plant-Microbe Interactions. 1993, 6: 565-572. 571 https://doi.org/10.1094/MPMI-6-565. 572 13. Ewels P, Magnusson M, Lundin S and Kaller M. MultiQC: summarize analysis 573 results for multiple tools and samples in a single report. Bioinformatics. 2016, 574 32(19):3047-3048. https://doi.org/10.1093/bioinformatics/btw354. 575 14. Flores-de la Rosa FR, De Luna E, Adame-García J, Iglesias-Andreu LG, Luna- 576 Rodríguez M. Phylogenetic position and nucleotide diversity of Fusarium 577 oxysporum f. sp. vanillae worldwide based on translation elongation 1α factor 578 sequences. Plant Pathology. 2018, 67(6): 1278-1285. 579 https://doi.org/10.1111/ppa.12847. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

580 15. Fravel D, Olivain C and Alabouvette C. Fusarium oxysporum and its biocontrol. 581 New Phytologist. 2003, 157(3): 493-502. https://doi.org/10.1046/j.1469- 582 8137.2003.00700.x. 583 16. Greene GH, McGary KL, Rokas A, Slot JC. Ecology Drives the Distribution of 584 Specialized Tyrosine Metabolism Modules in Fungi, Genome Biology and 585 Evolution. 2014, Volume 6, Issue 1, Pages 121–132, 586 https://doi.org/10.1093/gbe/evt208 587 17. Griffin, G. Exogenous carbon, and nitrogen requirements for chlamydospore 588 germination by Fusarium solani: dependence on spore density. Canadian Journal of 589 Microbiology. 1970, 16(12): 1366–1368. https://doi.org/10.1139/m70-226. 590 18. Griffin, G. Carbon, and nitrogen requirements for macroconidial germination of 591 Fusarium solani: dependence on conidial density. Canadian Journal of 592 Microbiology. 1970, 16(8): 733–740. https://doi.org/10.1139/m70-125. 593 19. Groenewald S. Biology, pathogenicity, and diversity of Fusarium oxysporum f.sp. 594 cubense. Master of Science dissertation. Pretoria University; 2006. 595 20. Howe K, Contreras-Moreira B, De Silva N, Maslen G, Akanni W, Allen J, Alvarez- 596 Jarreta J, Barba J, Bolser D, Cambell L, Carbajo M, Chakiachvili M, Christensen 597 M, Cummins C, Cuzick A, Davis P, Fexova S, Gall A, George N, Gil L, Gupta P, 598 Hammond-Kosack K, Haskell E, Hunt S, Jaiswal P, Janacek S, Kersey P, Langridge 599 N, Maheswari U, Maurel T, McDowall M, Moore B, Muffato M, Naamati G, 600 Naithani S, Olson A, Papatheodorou I, Patricio M, Paulini M, Pedro H, Perry E, 601 Preece J, Rosello M, Russell M, Sitnik V, Staines D, Stein J, Tello-Ruiz M, 602 Trevanion S, Urban M, Wei S, Ware D, Williams G, Yates A and Flicek 603 P. Ensembl Genomes 2020-enabling non-vertebrate genomic research. Nucleic 604 Acids Research. 2020, 48(D1): D689–D695. https://doi.org/10.1093/nar/gkz890. 605 21. Jastrzębowska K and Gabriel I. Inhibitors of amino acids biosynthesis as antifungal 606 agents. Amino Acids. 2015, 47(2):227-249. https://doi.org/10.1007/s00726-014- 607 1873-1. 608 22. Jones P, Binns D, Chang H, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, 609 Mitchell A, Nuka G, Pesseat S, Quinn A, Sangrador-Vegas A, Scheremetjew M, 610 Yong S, Lopez R and Hunter S. InterProScan 5: genome-scale protein function 611 classification. Bioinformatics. 2014, 30(9): 1236-1240. 612 https://doi.org/10.1093/bioinformatics/btu031. 613 23. Jonkers W, Rodrigues CD, Rep M. Impaired colonization, and infection of tomato 614 roots by the Deltafrp1 mutant of Fusarium oxysporum correlates with reduced 615 CWDE gene expression. Mol Plant Microbe Interact. 2009 May;22(5):507-18. doi: 616 10.1094/MPMI-22-5-0507. PMID: 19348569. 617 24. Joshi, S., Mathur, J.M.S. The positional distribution of fatty acids in phospholipids 618 of Fusarium oxysporum. Folia Microbiol. 1987, 32, 314–319. 619 https://doi.org/10.1007/BF02877219 620 25. Khalid AR, Zhang S, Luo X, Mehmood K, Rahim J, Shaheen H, Dong P, Qiu D, 621 Ren M. Role of Autophagy-Related Gene atg22 in Developmental Process and 622 Virulence of Fusarium oxysporum. Genes. 2019, 10(5):365. 623 https://doi.org/10.3390/genes10050365 624 26. Kanehisa M and Goto S. KEGG: Kyoto encyclopedia of genes and genomes. 625 Nucleic Acids Research. 2000, 28(1):27–30. https://doi.org/10.1093/nar/28.1.27. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

626 27. Kasote, D.M., Jayaprakasha, G.K., Singh, J. et al. Metabolomics-based biomarkers 627 of Fusarium wilt disease in watermelon plants. J Plant Dis Prot. 2020, 127, 591– 628 596. https://doi.org/10.1007/s41348-020-00314-0 629 28. Kihara A and Igarashi Y. Cross talk between sphingolipids and 630 glycerophospholipids in the establishment of plasma membrane asymmetry. Mol. 631 Biol. Cell. 2010, 15, 4949–4959. doi: 10.1091/mbc. E04-06-0458 632 29. Kong X, Zhang H, Wang X, van der Lee T, Waalwijk C, van Diepeningen A, 633 Brankovics B, Xu J, Xu J, Chen W and Feng J. FgPex3, a Peroxisome Biogenesis 634 Factor, Is Involved in Regulating Vegetative Growth, Conidiation, Sexual 635 Development, and Virulence in Fusarium graminearum. Front. Microbiol. 2019, 636 10:2088. doi: 10.3389/fmicb.2019.02088 637 30. Koyyappurath S, Conéjéro G, Dijoux J, Lapeyre-Montés F, Jade K, Chiroleu F, 638 Gitaneae F, Verdeil J, Besse P and Grisoni M. Differential responses of Vanilla 639 accessions to root rot and colonization by Fusarium oxysporum f. sp. radicis- 640 vanillae. Frontiers in Plant Science. 2015, 6: 1-16. 641 https://doi.org/10.3389/fpls.2015.01125. 642 31. Koyyappurath S, Atuahiva T, Guen L, Batina H, Le Squin S, Gautheron N, 643 Hermann V, Peribe J, Jahiel M, Steinberg C, Liew E, Alabouvette C, Besse P, Dron 644 M, Sache I, Laval V and Grisoni M. Fusarium oxysporum f. sp. radicis-vanillae is 645 the causal agent of root and stem rot in vanilla. Plant Pathology. 2016, 65(4): 612- 646 625. https://doi.org/10.1111/ppa.12445. 647 32. Kumar Y, Zhang L, Panigrahi P, Dholakia BB, Dewangan V, Chavan SG, Kunjir 648 SM, Wu X, Li N, Rajmohanan PR, Kadoo NY, Giri AP, Tang H, Gupta VS. 649 Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots 650 as revealed by a combination of proteomics and metabolomics. Plant Biotechnol J. 651 2016, 14(7):1589-603. doi: 10.1111/pbi.12522. Epub 2016 Jan 23. PMID: 652 26801007; PMCID: PMC5066658. 653 33. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G 654 and Durbin R. The Sequence Alignment/Map format and SAMtools. 655 Bioinformatics. 2009, 25(16): 2078-2079. 656 https://doi.org/10.1093/bioinformatics/btp352. 657 34. Liu TB, Xue C. The Ubiquitin-Proteasome System and F-box Proteins in 658 Pathogenic Fungi. Mycobiology. 2011, 39(4):243-8. doi: 659 10.5941/MYCO.2011.39.4.243. Epub 2011 Dec 7. PMID: 22783111; PMCID: 660 PMC3385136. 661 35. Liew E, Pinaria A, Rondonuwu F, Paath J, Sembel D and Burgess L. Vanilla stem 662 rot pathogen can survive as an endophyte within healthy vines. Journal of Plant 663 Pathology. 2008, 90: 414. 664 36. Lucas J. Plant Pathology and Plant Pathogens 3rd Edition. New York, USA: Wiley- 665 BlackWell; 1998. 666 37. Lv W, Wang C, Yang N, Que Y, Talbot NJ, Wang Z. Genome-wide functional 667 analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol 668 production and virulence in Fusarium graminearum. 2017, Sep 11;7(1):11062. doi: 669 10.1038/s41598-017-11640-z. PMID: 28894236; PMCID: PMC5594004. 670 38. Ma L, Van der Does C, Borkovich K, Coleman J, Daboussi M, Di Pietro A, 671 Dufresne M, Freitag M, Grabherr M, Henrissat B, Houterman P, Kang S, Shim W, 672 Woloshuk C, Xie X, Xu J, Antoniw J, Baker S, Bluhm B, Breakspear A, Brown D, bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

673 Butchko R, Chapman S, Coulson R, Coutinho P, Danchin E, Diener A, Gale L, 674 Gardiner D, Goff S, Hammond-Kosack K, Hilburn K, Hua-Van A, Jonkers W, 675 Kazan K, Kodira C, Koehrsen M, Kumar L, Lee Y, Li L, Manners J, Miranda- 676 Saavedra D, Mukherjee M, Park G, Park J, Park S, Proctor R, Regev A, Ruiz- 677 Roldan M, Sain D, Sakthikumar S, Sykes S, Schwartz D, Turgeon B, Wapinski I, 678 Yoder O, Young S, Zeng Q, Zhou S, Galagan J, Cuomo C, Kistler H and Rep M. 679 Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. 680 2010, 464: 367-373. https://doi.org/10.1038/nature08850. 681 39. Ma L, Geiser D, Proctor R, Rooney A, O’Donnell K, Trail F, Gardiner D, Manners 682 J and Kazan K. Fusarium Pathogenomics. Annual Reviews. 2013, 67: 399-416. 683 https://doi.org/10.1146/annurev-micro-092412-155650 684 40. MacHardy W and Beckman C. Vascular wilt Fusaria: infection and pathogenesis in 685 fusarium: diseases, biology, and taxonomy. In: Nelson P. E., Toussoun T. A., Cook 686 R. J. editors. Pennsylvania, USA: The Pennsylvania State University Press; 1981. p. 687 365–391. 688 41. Manikandan R, Harish S, Karthikeyan G, Raguchander T. Comparative Proteomic 689 Analysis of Different Isolates of Fusarium oxysporum f.sp. lycopersici to Exploit 690 the Differentially Expressed Proteins Responsible for Virulence on Tomato Plants. 691 Frontiers in Microbiology. 2018, 9:420. DOI: 10.3389/fmicb.2018.00420 692 42. Michielse, C. B., and Rep, M. Pathogen profile update: Fusarium oxysporum. Mol. 693 Plant Pathol. 2009, 10, 311–324. doi: 10.1111/j.1364-3703.2009. 00538.x 694 43. Miguel-Rojas C, Hera C. Proteomic identification of potential target proteins 695 regulated by the SCF(F) (bp1) -mediated proteolysis pathway in Fusarium 696 oxysporum. Molecular Plant Pathology. 2013, Dec;14(9):934-945. DOI: 697 10.1111/mpp.12060. 698 44. Nebauer, R., Birner-Grünberger, R., and Daum, G. 3 Biogenesis and cellular 699 dynamics of glycerophospholipids in the yeast Saccharomyces cerevisiae, in Lipid 700 Metabolism and Membrane Biogenesis. Topics in Current Genetics. 2004, Vol. 6, 701 ed. G. Daum (Berlin: Springer), 125–168. doi: 10.1007/978-3-540-40999-1_4 702 45. Okada T, & Matsubara Y. Tolerance to Fusarium Root Rot and the Changes in Free 703 Amino Acid Contents in Mycorrhizal Asparagus Plants, HortScience horts. 2012, 704 47(6), 751-754. Retrieved Mar 21, 2021, from 705 https://journals.ashs.org/hortsci/view/journals/hortsci/47/6/article-p751.xml 706 46. Olivain C and Alabouvette C. Colonization of tomato root by a non-pathogenic 707 strain of Fusarium oxysporum. New Phytologist. 1997, 137:481-494. 708 https://doi.org/10.1046/j.1469-8137.1997.00855.x. 709 47. Olivain C and Alabouvette C. Process of tomato root colonization by a pathogenic 710 strain of Fusarium oxysporum f. sp. Lycopersici in comparison with a non- 711 pathogenic strain. New Phytologist. 1999, 141: 497-510. 712 https://doi.org/10.1046/j.1469-8137.1999.00365.x. 713 48. Olivain C, Humbert C, Nahalkova J, Fatehi J, L´Haridon F. and Alabouvette, C. 714 Colonization of tomato root by pathogenic and nonpathogenic Fusarium oxysporum 715 strains inoculated together and separately into the soil. Applied and Environmental 716 Microbiology. 2006, 72(2): 1523-1531. https://doi.org/10.1128/AEM.72.2.1523- 717 1531.2006. 718 49. O'Donnell K, Kistler H, Cigelnik E and Ploetz R. Multiple evolutionary origins of 719 the fungus causing Panama disease of banana: Concordant evidence from nuclear bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

720 and mitochondrial gene genealogies. PNAS. 1998, 95(5): 2044-2049. 721 https://doi.org/10.1073/pnas.95.5.2044. 722 50. Phillips DA, Fox TC, King MD, Bhuvaneswari TV, Teuber LR. Microbial Products 723 Trigger Amino Acid Exudation from Plant Roots. Plant Physiology. 2004, 136 (1) 724 2887-2894; DOI: 10.1104/pp.104.044222 725 51. Pinaria A, Liew E and Burgess L. Fusarium species associated with vanilla stem rot 726 in Indonesia. Australasian Plant Pathology. 2010, 39: 176-183. 727 https://doi.org/10.1071/AP09079. 728 52. Pinaria A, Laurence M, Burgess L and Liew E. Phylogeny and origin of Fusarium 729 oxysporum f. sp. vanillae in Indonesia. Plant Pathology. 2015, 64(6): 1358-1365. 730 https://doi.org/10.1111/ppa.12365. 731 53. Quinlan A and Hall I. BEDTools: a flexible suite of utilities for comparing genomic 732 features. Bioinformatics. 2010, 26(6): 841-842. 733 https://doi.org/10.1093/bioinformatics/btq033. 734 54. Scandiani MM, Luque AG, Razori MV, Ciancio Casalini L, Aoki T, O'Donnell K, 735 Cervigni GDL, Spampinato CP. Metabolic profiles of soybean roots during early 736 stages of Fusarium tucumaniae infection, Journal of Experimental Botany. 2015, 737 Volume 66, Issue 1, Pages 391–402, https://doi.org/10.1093/jxb/eru432 738 55. Sharma, M., Sengupta, A., Ghosh, R. et al. Genome wide transcriptome profiling of 739 Fusarium oxysporum f sp. ciceris conidial germination reveals new insights into 740 infection-related genes. 2016, Sci Rep 6, 37353. https://doi.org/10.1038/srep37353 741 56. Solano-De la Cruz M, Adame-Garcia J, Gregorio-Jorge J, Jimenez-Jacinto V, Vega- 742 Alvarado L, Iglesias-Andreu L, Escobar-Hernandez, E and Luna-Rodriguez, M. 743 Functional categorization of de novo transcriptome assembly of Vanilla planifolia 744 Jacks. potentially points to a translational regulation during early stages of infection 745 by Fusarium oxysporum f. sp. vanillae. BMC Genomics. 2019, 20 (826): 1-15. 746 https://doi.org/10.1186/s12864-019-6229-5. 747 57. Schneider E and Carlquist S. Pit membrane remnants in perforation plates and other 748 vessel details of cornales. Brittonia. 2004, 56: 275-283. 749 https://doi.org/10.1663/0007-196X(2004)056[0275:PMRIPP]2.0.CO;2. 750 58. Taylor J, Geiser D, Burt A and Koufopanou V. The Evolutionary Biology and 751 Population Genetics Underlying Fungal Strain Typing. Clinical Microbiology 752 Reviews. 1999, 12(1): 126-146. https://doi.org/10.1128/CMR.12.1.126. 753 59. Taylor J, Jacobson D and Fisher M. The evolution of asexual fungi: reproduction, 754 speciation, and classification. 1999, 37: 197-246. 755 https://doi.org/10.1146/annurev.phyto.37.1.197. 756 60. Tchameni NS, Nwaga D, Nana WL, Ngonkeu MEL. Fokom R. Etoa F.X. Growth 757 enhancement, amino acid synthesis and reduction in susceptibility towards 758 Phytophthora megakarya by Arbuscular Mycorrhizal Fungi inoculation in cocoa 759 plants. J. Phytopathol. 2012; https://doi.org/10.1111/j.1439-0434.2012.01888.x 760 61. Tombe M, Kobayashi K and Ogoshi A. Vegetative compatibility grouping of 761 Fusarium oxysporum f. sp. vanillae in Indonesia. Indonesian Journal of Crop 762 Science. 1994, 9: 29–39. 763 62. Thomas J, Vijayan A and Bhai R. Vanilla disease in India and their management. 764 Indian Journal of Arecanut Spices and Medical Plants. 2002, 4: 143–149. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

765 63. van Aarle IM, Olsson PA. Fungal lipid accumulation and development of mycelial 766 structures by two arbuscular mycorrhizal fungi. Appl Environ Microbiol. 2003, 69: 767 6762–6767 768 64. Wang H, Wang Z, Liu Z, Wang K, Xu W. Membrane disruption of Fusarium 769 oxysporum f. sp. niveum induced by myriocin from Bacillus amyloliquefaciens 770 LZN01. Microb Biotechnol. 2020, https://doi.org/10.1111/1751-7915.13659. 771 65. Yu G, Wang L, Han Y and He Q. clusterProfiler: An R Package for Comparing 772 Biological Themes Among Gene Clusters. OMICS. 2012, 16(5): 284-287. 773 https://doi.org/10.1089/omi.2011.0118. 774 66. Zuo C, Zhang W, Chen Z, Chen B and Huang Y. RNA Sequencing Reveals that 775 Endoplasmic Reticulum Stress and Disruption of Membrane Integrity Underlie 776 Dimethyl Trisulfide Toxicity against Fusarium oxysporum f. sp. cubense Tropical 777 Race 4. Front. Microbiol. 2017, 8:1365. doi: 10.3389/fmicb.2017.01365 778 67. Zhang, X., Wang, H., Zhu, W. et al. Transcriptome Analysis Reveals the Effects of 779 Chinese Chive (Allium tuberosum R.) Extract on Fusarium oxysporum f. sp. 780 radicis-lycopersici Spore Germination. Curr Microbiol. 2020, 77, 855–864. 781 https://doi.org/10.1007/s00284-020-01875-x

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798 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

799 Figures and Tables

Dataset to filter Number Dataset used as Number Generated Number of Number of of genes filter of genes dataset genes genes obtained filtered 2 dpi treatment 6174 2 dpi control 5455 Genes involved in 3045 3129 infection at 2 days 10 dpi treatment 13092 10 dpi control 5765 Genes involved in 7734 5358 infection at 10 days Genes involved in 3045 Genes involved in 7734 Unique genes of 1334 1711 infection at 2 days infection at 10 early infection days Genes involved in 7734 Genes involved in 3045 Unique genes of 6023 1711 infection at 10 infection at 2 days late infection days 800 Table 1. Summary of the filter analysis of the reads aligned with the reference genome of 801 Fusarium oxysporum f. sp. vanillae Fol 4287, contrasting the transcripts present in the 802 treatment libraries with the JAGH3 strain vs the controls and at 2 dpi and 10 dpi.

803

Category Gene Count

Early stage infection 3045

Late stage infection 7734

Exclusive of early stage infection 1334

Exclusive of late stage infection 6023

804 Table 2. Shared and not shared genes numbers between early and late stage of infection.

805

806

807

808

809

810

811

812

813

814

815 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Gene ID Coding protein Fusarium species

FOXG_15434 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_05948 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_09893 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_09744 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13795 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_08862 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_12264 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_02513 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13794 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_04765 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_11739 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_14695 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13051 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_12330 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_12863 Hypervirulence associated protein, TUDOR domain F. oxysporum f. sp. vanillae

FOXG_15415 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_09894 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

816 Table 3. Hypervirulence and virulence factors of Fov at 2 dpi in V. planifolia Jacks.

817

818

819

820

821

822

823

824

825 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Gene ID Coding protein Fusarium species

FOXG_17741 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_15434 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_05948 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_05861 Egh16-like virulence factor F. oxysporum f. sp. vanillae

FOXG_13331 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_09893 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_11096 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13795 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_08862 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_12264 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_02509 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13794 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_11739 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_05407 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_16516 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13051 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13801 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_06038 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_12863 Hypervirulence associated protein, TUDOR domain F. oxysporum f. sp. vanillae

FOXG_10052 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_15415 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_11740 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_14660 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_13103 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_09744 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_02712 Egh16-like virulence factor F. oxysporum f. sp. vanillae

FOXG_02513 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_16409 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_14695 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_10728 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

FOXG_12330 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_10062 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_17000 Pectin lyase fold/virulence factor F. oxysporum f. sp. vanillae

FOXG_10173 Egh16-like virulence factor F. oxysporum f. sp. vanillae

826 Table 4. Hypervirulence and virulence factors of Fov at 10 dpi in V. planifolia Jacks.

827

Gene ID Coding protein Fusarium species

FOXG_09277 Stage II sporulation protein E (SpoIIE) F. oxysporum f. sp. vanillae

FOXG_08691 Sporulation-specific protein Spo7 F. oxysporum f. sp. vanillae

828 Table 5. Sporulation genes of Fov at 2 dpi in V. planifolia Jacks.

829

Gene ID Coding protein Fusarium species

FOXG_09418 Clp amino terminal domain, pathogenicity island component F. oxysporum f. sp. vanillae

FOXG_07723 Necrosis inducing protein (NPP1) F. oxysporum f. sp. vanillae

830 Table 6. Necrosis/pathogenic genes of Fov at 2 dpi in V. planifolia Jacks.

831

Gene ID Coding protein Fusarium species

FOXG_09823 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_08593 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_16912 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

832 Table 7. Fusaric acid related genes of Fov at 2 dpi in V. planifolia Jacks.

833

Gene ID Coding protein Fusarium species

FOXG_09277 Stage II sporulation protein E (SpoIIE) F. oxysporum f. sp. vanillae

FOXG_05170 Conidiation protein 6 F. oxysporum f. sp. vanillae

FOXG_08691 Sporulation-specific protein Spo7 F. oxysporum f. sp. vanillae

834 Table 8. Conidiation and sporulation genes of Fov at 10 dpi in V. planifolia Jacks.

835 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Gene ID Coding protein Fusarium species

FOXG_06413 Pathogen effector; putative necrosis-inducing factor F. oxysporum f. sp. vanillae

FOXG_09418 Clp amino terminal domain, pathogenicity island component F. oxysporum f. sp. vanillae

FOXG_17014 Necrosis inducing protein (NPP1) F. oxysporum f. sp. vanillae

FOXG_07723 Necrosis inducing protein (NPP1) F. oxysporum f. sp. vanillae

836 Table 9. Necrosis/pathogenic genes of Fov at 10 dpi in V. planifolia Jacks.

837

Gene ID Coding protein Fusarium species

FOXG_09823 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_08869 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_16912 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_08593 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_06206 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_05525 Fusaric acid resistance protein-like F. oxysporum f. sp. vanillae

FOXG_09319 Fusarinine C esterase SidJ F. oxysporum f. sp. vanillae

838 Table 10. Fusarinine and fusaric acid related genes of Fov at 10 dpi in V. planifolia Jacks.

839

840

841

842

843

844

845

846

847

848 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

849 850 Figure 1. Fol 4287 karyotype with alignment of JAGH3 strain transcripts present 851 during vanilla infection at 2 dpi. A) the transcripts corresponding to the control treatment. 852 B) transcripts present in the treatment with the pathogenic strain JAGH3. C) intersection of 853 the transcripts presents in the control treatment and the treatment with the JAGH3 strain.

854

855

856

857 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

858 859 Figure 2. Fol 4287 karyotype with alignment of JAGH3 strain transcripts present 860 during vanilla infection at 10 dpi. A) the transcripts corresponding to the control 861 treatment. B) transcripts present in the treatment with the pathogenic strain JAGH3. C) 862 intersection of the transcripts presents in the control treatment and the treatment with the 863 JAGH3 strain.

864

865

866 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

867 868 Figure 3. Gene Ontology of top 30 biological processes of Fov at 2 dpi in V. planifolia 869 Jacks.

870

871

872

873

874 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

875 876 Figure 4. Gene Ontology of top 30 biological processes of Fov at 10 dpi in V. planifolia 877 Jacks.

878

879

880

881

882 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

883 884

885 Figure 5. KEGG Gene Enrichment Analysis of top 20 metabolic pathways of Fov at 2 dpi 886 in V. planifolia Jacks.

887

888

889

890

891

892

893

894

895

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897

898

899 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

900 901 Figure 6. KEGG Gene Enrichment Analysis of top 30 metabolic pathways of Fov at 10 dpi 902 in V. planifolia Jacks.

903

904

905

906

907 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436347; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

908 909 Figure 7. Hypothetical model of the Fov pathogenesis.

910

911 Supplementary material

912

913

914 915 Supplementary Figure 1. Track files visualization of the treatment strain JAGH3 (red 916 tracks) at 10 dpi, its control (blue tracks) and the gene structure below in the genomic 917 coordinates corresponding to a Pathogenicity Island Component in IGV Genome Browser.

918 Supplementary Files

919 2_dpi_treatment_genes_list.txt

920 10_dpi_treatment_genes_list.txt

921 Metabolic_routes_of_early_infection_at_2_dpi.xlsx

922 All_the_metabolic_routes_of_the_infection_dead_10_days.xlsx