Computational proteomics analysis reveals pathogen secreted carbohydrate active enzymes in tomato xylem sap

Fusarium oxysporum f. sp. lycopersici (Fol), a causal organism of Fusarium wilt in the tomato plant, secretes cell wall degrading enzymes, also known as carbohydrate-active enzymes (CAZymes). These are crucial during colonization and pathogenesis, as evidenced by several proteomic studies, revealing the importance of these CAZymes in virulence and pathogenicity. However, few of them have been done in-planta, exhibiting differences in the expression of these cell wall degrading enzymes compared to in-vitro studies. Therefore, to explore the CAZymes involved in pathogenesis while residing in the host plant, an in-planta (xylem sap) proteomics of a susceptible tomato variety affected with Fol was done. Most of these CAZymes belonged to the hydrolase and oxidoreductase families having no signicant homology with tomato proteins. Nearly 90% of them were predicted to be soluble and extracellular. The core CAZymes families with interactional evidence identied were AA3, GH3, GH18, GH20, GH28, GH43, GH47, GH55 and CE8. Thus, apart from annotating some of the hypothetical proteins to be CAZymes, the study sheds light on CAZymes families that may have a role in the pathogenesis and survival of this fungus in the susceptible tomato plant. study includes a differential analysis of a resistant and susceptible tomato variety infected with F.oxysporum f. sp. lycopersici (Fol). Samples consisting of a susceptible host infected with Fol and a mock treated of the same tomato cultivar were taken for further analysis, based on the hypothesis proposed in the current study. A total of eight samples, i.e., four replicates each (FR24 to FR27) considered as treated (T) and (FR12 to FR15) considered as mock treated (M) in PXD011072 were taken. These experiments were done in a climate controlled greenhouse at 25 º C with a relative humidity of 65% and 16 hours photoperiod. The pathogen used was Fol race 1 (fol007) and the susceptible tomato plant cultivar C32. Protein sequences of the organisms Tomato (Solanum lycopersicum assembly SL3.0) consisting of 37660 proteins and Fusarium oxysporum (Fusarium oxysporum f. sp. lycopersici 4287 assembly ASM14995v2) consisting of 27347 proteins were downloaded from NCBI database.


Introduction
Fusarium oxysporum species complex (FOSC) causing wilt and rot disease in various plant species is generally host-speci c and known as formae speciales, ff. spp. (singular formae specialis, f. sp.). These plant pathogenic formae speciales follow a similar infection mechanism in the host plant, which includes penetration through root and colonization in the xylary tract (Lagopodi et al. 2002;Jiménez-Fernández et al. 2013;Lu et al. 2013;Upasani et al. 2016;Dong et al. 2019), resulting in wilting in some cases, root rot of susceptible plants leading to necrosis. One such formae specialis is f. sp. lycopersici, affecting the important vegetable crop tomato grown worldwide (http://www.fao.org/faostat/en/#data/QC/visualize).
To penetrate the root for successful colonization, f. sp. lycopersici requires crossing the cell wall barrier of the host plant. These cell wall barriers are composed of cellulose, hemicellulose, pectin, lignin, and other proteins, essentially present for structural integrity (Rose et al. 2004;Cosgrove 2005). The process of cell wall deconstruction by the pathogen is achieved by the enzymes known as plant cell wall degrading enzymes (PCWDE), which within the host plants help degrade the plant tissues for liberating mono-and oligosaccharides required for their growth (Kubicek et al. 2014). Some of these enzymes are also needed for cell wall remodeling (Beauvais and Latgé 2018) and at the later stage of colonization (Gibson et al. 2011). These CWDEs are a subset of enzyme families know as Carbohydrate active enzymes (CAZymes), which according to the CAZy database (http://www.cazy.org/) (Lombard et al. 2014), are divided into ve modules, i.e., Auxiliary activities (AA), carbohydrate Esterase (CE), Glycosyl Hydrolase (GH), Glycosyl Transferase (GT), Polysaccharide Lyase (PL) and one associated module, Carbohydrate-Binding Modules (CBM), consisting of several families based on functional, structural and sequence similarities. The families in these modules are further divided into four broad categories based on their activities, such as cellulose-degrading (mostly GH family), hemicellulose degrading (GH and CE), pectin (GH, CE, and PL) and lignin-degrading (mostly AA). In contrast, members of the GT family are involved in the biosynthetic process. The FOSC and other sordariomycetes harbor a plethora of CAZymes in their genome (https://mycocosm.jgi.doe.gov/mycocosm/annotations/browser/cazy/summary;85ATbn?p=dothideomycetes) with a majority of them belonging to GH families followed by GT, AA, CE, PL and being necrotrophic, codes for a signi cant amount of PCWDE as well as secretory CAZymes in Fusarium oxysporum formae speciales (Roy et al. 2020).
Over the last decade, fungal proteomics have taken the lead in the eld of omics due to the availability of full-length fungal genomes, high throughput transcriptomic data and advancements in protein mass spectrometry, which gave us a valuable insight into the pathogenicity of the plant fungal pathogens.
Although proteomics analysis of host plants in response to Fusarium oxyporum formae speciales, such as banana (Li et al. 2013;Lu et al. 2013;Dong et al. 2019), cabbage (Pu et al. 2016), cucumber (Zhang et al. 2016), pea (Castillejo et al. 2015), tomato (Gawehns et al. 2015;Silva et al. 2017;de Lamo et al. 2018) and strawberry (Fang et al. 2013) have been done, but several proteomics studies were also performed in Fusarium species, including the Fusarium oxysporum ff. spp. For instance, an exoproteome study on Fusarium graminearum grown on the cell wall of Humulus lupulus L. (Phalip et al. 2005) revealed Cellobiohydrolase (belonging to GH6 and GH48) to be pre-dominant in pathogen-plant interaction. Similarly, in a study (Paper et al. 2007) on the same fungal species, in-planta and in-vitro analyses evidenced the presence of hydrolase, lyase, and esterase in the former, suggesting their role in pathogenesis. Likewise, another study on Fusarium proliferatum also showed inhibition of CWDEs consisting of glucanase, cellulase, and glucanosyltransferase (Li et al. 2017) to be in uenced by high pH, therefore affecting its growth.
Among the F. oxysporum ff spp., an in vitro comparative proteomics studies on the races of f. sp. cubense and f. sp. conglutinans enabled the identi cation of proteins involved in carbohydrate metabolism, in which chitinase and α-galactosidase were revealed to be differentiating the virulence pattern in former (Sun et al. 2014), whereas the glycosidase protein was observed to differ among the races of the latter (Li et al. 2015). Moreover, some CWDE in f. sp. cubense, such as glucanase and cellobiose dehydrogenase, were found to be downregulated by deleting a transcription factor resulting in the reduction of the pathogenicity (Hou et al. 2018). Simultaneously, some cell wall modifying enzymes were also expressed during conidial germination (Deng et al. 2015), depicting their role in growth. Likewise, another comparative proteomics analysis of different isolates of f. sp. lycopersici revealed the importance of endo-β-Nacetylglucosaminidases (GH85) in imparting the virulence (Manikandan et al. 2018). Although most proteomics work is done so far to identify and decipher the role of cell wall degrading enzymes has been through in-vitro studies. Still, very few of them were reported in-planta using Fusarium species. Concerning the formae speciales, any study reported so far was only on f. sp. lycopersici assessing the pathogenicity of chromosomes (Schmidt et al. 2013). Thus, to narrow down this gap with the process of annotating and implicating the role of CAZymes in pathogenesis, a downstream proteomics analysis was done using publically available proteomics data of tomato xylem sap infected with Fusarium oxysporum f. sp. lycopersici. Further, using computational tools to substantiate the importance of these cell wall degrading enzymes which would otherwise may not be expressed at the protein level under in-vitro conditions in this pathogenic fungus.

Data retrieval
The LTQ Orbitrap label-free mass spectrometric publically available data of tomato xylem sap with accession: PXD011072 (https://www.ebi.ac.uk/pride/archive/projects/PXD011072) was downloaded from PRIDE (https://www.ebi.ac.uk/pride/) which constituted the experiments performed elsewhere (Gawehns et al. 2015;de Lamo et al. 2018). This study includes a differential proteomics analysis of a resistant and susceptible tomato variety infected with F.oxysporum f. sp. lycopersici (Fol). Samples consisting of a susceptible host infected with Fol and a mock treated of the same tomato cultivar were taken for further analysis, based on the hypothesis proposed in the current study. A total of eight samples, i.e., four replicates each (FR24 to FR27) considered as treated (T) and (FR12 to FR15) considered as mock treated (M) in PXD011072 were taken. These experiments were done in a climate controlled greenhouse at 25 º C with a relative humidity of 65% and 16 hours photoperiod. The pathogen used was Fol race 1 (fol007) and the susceptible tomato plant cultivar C32. Protein sequences of the organisms Tomato (Solanum lycopersicum assembly SL3.0) consisting of 37660 proteins and Fusarium oxysporum (Fusarium oxysporum f. sp. lycopersici 4287 assembly ASM14995v2) consisting of 27347 proteins were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/) database.

Data processing
All the raw les of a susceptible plant treated with Fol and mock treated were processed in the MaxQuant version 1.6.17.0 (Tyanova et al. 2016a) for protein identi cation using an inbuilt search engine against the protein database created by taking Fol and tomato proteins mentioned above with an in-built contaminant database (246 proteins). Label-free quanti cation was done using default parameters (Cox et al. 2011(Cox et al. , 2014 with a match between runs enabled and label-free quanti cation (LFQ) minimum ratio count set to 1. An extra variable modi cation-deamidation (NQ), performed by original authors (de Lamo et al. 2018) was also included for the protein quanti cation. The data generated were further ltered and analyzed on Perseus 1.6.14.0 (Tyanova et al. 2016b), where all the peptide counts less than 2 identifying a protein group were ltered out. The LFQ intensities were further Log 2 (x) transformed to visualize the data. The proteins identi ed in the tomato were removed for subsequent analysis retaining the proteins identi ed by at least one unique and one unmodi ed peptide for the Fol proteins.

Gene Ontology (GO) analysis
The Fol proteins identi ed in the xylem sap were subjected to gene ontologies (GO) annotation using Blast2GO 5.2.5 suite (Götz et al. 2008) with an in-built InterPro tool by keeping default settings to see the categorical representation of these proteins in individual studies. The proteins with GO annotations were further visualized using WEGO 2.0 (http://wego.genomics.org.cn/) (Ye et al. 2018) for the Fol proteins identi ed in the experiment. Additionally, GO term enrichment analysis was also performed to visualize the functional enrichment using web-based tool g: Pro ler (https://biit.cs.ut.ee/gpro ler/gost) (Raudvere et al. 2019) after selecting the organism Fusarium oxysporum f. sp. lycopersici and with the default options for the enrichment process.

Carbohydrate-active enzymes (CAZymes) annotation
A web-based meta server as well as a stand-alone tool-dbCAN2 (http://bcb.unl.edu/dbCAN2/index.php) ) was used to annotate the CAZymes among the identi ed protein groups in Fol. This web-based meta server annotates CAZymes using three integrated search tools, HMMER (http://hmmer.org/), DIAMOND (https://github.com/bbuch nk/diamond) and HotPep (Busk et al. 2017) for the annotation of CAZymes with their default parameters from the search databases viz. dbCAN, CAZy and PPR databases, respectively. This meta server tool suggests retaining only those CAZymes, which are annotated with a minimum of 2 integrated tools for better prediction con dence.

Secretory protein and subcellular localization analysis
As the xylem sap collected in these datasets were devoid of any physical presence of fungus. Therefore, to ascertain if CAZymes annotated in the identi ed Fol proteins were secretory or extracellular in origin, two online tools, namely, SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/) (Almagro Armenteros et al. 2019) and DeepLoc 1.0 (http://www.cbs.dtu.dk/services/DeepLoc/) (Almagro Armenteros et al. 2017), were used. These two tools work on deep-and recurrent-neural network approaches, respectively. The former predicted the signal peptides by keeping the organism group-Eukarya, and the latter identi ed the subcellular localization by keeping PROFILES as a protein-coding option for accurate prediction.

Metabolic pathway enrichment and ortholog interaction network analysis
The CAZymes identi ed were analyzed for their chromosomal location by taking the respective gene accessions of the identi ed CAzymes through the genome view option and metabolic pathways enrichment was also done with a p-value <0.05 using FungiDB (https://fungidb.org/fungidb/app) (Basenko et al. 2018). Further, an evidence-based protein interaction network was analyzed using STRING 11.0 (https://string-db.org/) (Szklarczyk et al. 2019) by taking clusters of orthologous groups (COG), Eukaryotic orthologous group (KOG) and non-supervised orthologous groups (NOG) for the corresponding CAZymes families with the settings: minimum required interaction score of 0.700 (high con dence) and a maximum number of ve interactors in the rst shell to see the existing interaction among these ortholog groups of the identi ed CAZymes.

Identi cation of protein groups and statistical analysis
The proteins identi ed in the individual sample dataset after ltering (i.e., the removal of contaminants, reverse hits, and proteins identi ed only by site) revealed the number of proteins identi ed in tomato and Fol is summarized in Table 1 Table 1). The histogram revealed the data to be more or less normally distributed. These replicates showed a Pearson correlation of more than 0.8 in both mock and treated samples (Fig. 2).

Functional assessment of Fol proteins
All the Fol proteins identi ed in the susceptible tomato plant have proceeded for GO annotation. A total of 36 out of 53 protein accessions were assigned for some GO terms. Among these GO terms, 32 GO terms were assigned for biological functions, whereas 36 GO terms were assigned to some molecular functions with 3 GO terms belonging to cellular functions. The histogram of GO categories revealed the majority of the GO categories were involved in primary and organic substance metabolic processes followed by the oxidation-reduction process with a function of hydrolase and oxidoreductase activity (Fig. 3a).
In gene enrichment analysis, the proteins in each experiment were assigned to their respective gene accessions. The enrichment analysis using g:SCS threshold, which is an algorithm developed by the authors of the tool utilized for multiple testing correction, revealed the enriched biological process to be carbohydrate metabolic process (GO: 0005975) and response to oxidative stress (GO: 0006979) with most signi cant molecular functions of hydrolase activity (GO: 0016798, GO: 0004553, GO:0016787), polygalacturonase activity (GO:0004650) catalase activity (GO:0004096), peroxidase activity (GO:0004601), oxidoreductase activity (GO:0016684), antioxidant activity (GO:0016209) and catalytic activity(GO:0003824) (Fig. 3b).

Annotated and core CAZymes in Fol proteins
After ltering the CAZymes annotated by >= 2 tools from dbCAN2 annotation results ( Fig. 4; Supplementary Table 2), altogether, 30 protein accessions were annotated as CAZymes. Most of the CAZymes belonged to the families of GH, followed by AA and only one CE family, i.e., CE8 (Table 2). Among these protein accessions annotated for CAZymes, 16 of them were hypothetical proteins (HP). Further, the GO annotation from the already annotated protein groups in the previous step revealed 24 protein accessions coding for CAZymes families were having GO terms associated with them (Supplementary Table 3). Interestingly, most GO categories were identi ed to be involved in primary and organic substance metabolic processes having hydrolase and oxidoreductase activities.

Secreted protein and subcellular localization of identi ed CAZymes
To con rm the CAZymes identi ed above belonged to secretory proteins and extracellular in function, which apparently may in uence the process of pathogenesis and colonization after infecting the host plant, they were subjected to secretory protein analysis. Interestingly, nearly 90% of the CAZymes were found to have standard signal peptides cleaved by signal peptidase I (Table 2, Supplementary Table 4 and 5) and extracellular in nature except for GH47 and GH72, though harboring signal peptide. Most of these extracellular CAZymes combined together were generally hemicellulose degrading (GH18, GH20, GH43, GH93, GH32), followed by cellulose/hemicellulose degradation (GH45, GH3), oxidoreductase (AA3, AA5) and pectin degrading (CE8, GH28).

Chromosomal location, metabolic pathway enrichment and interaction network of CAZymes
The CAZymes identi ed were found to be located on chromosome 1, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 and 14 (Table 3), with half of them to be located on the reverse strand. In the metabolic enrichment process, the pathway in which these CAZymes involved were pectin degradation (CE8 and GH28), choline degradation (AA3_2) and chitin degradation (GH18, GH20, GH45, GH47) with a p-value from Fisher's exact test, Bonferroni adjusted p-value n and Benjamini Hochberg false discovery rate (FDR) cut-off <0.05 (Supplementary Table 6). The CAZymes families were further studied for an evidence-based interactional network to elucidate a relationship among these CAZymes present in xylem sap of the host plant infected with pathogen Fol. Hence, an interaction network of ortholog groups, COG, KOG and NOG corresponding to the gene accessions of each protein group was created, which resulted in 7 CAZymes families encompassing 12 protein accessions (Table 4) having direct and indirect interaction within them. The interaction network with a combined interaction score ranged from 0.7-0.9 (Supplementary Table 7) and revealed six of the CAZymes families, i.e., GH3, GH18, GH20, GH28, GH43 and CE8, having direct interaction evidence. In contrast, AA3 linked with GH18 through an enzyme Acyl-CoA reductase (COG1012), GH3 linked with GH18 through pectate lyase (COG3866), GH32 and GH55 were connected through a glucanase (GH17 family, COG5309) whereas, GH74 was interlinked with a mannosidase (KOG2204) and a transferase (KOG1413), revealed to be involved in the polysaccharide degrading process (Fig. 5).

Discussion
The plant pathogen Fusarium oxysporum f. sp. lycopersici, a causal organism of tomato wilt, like many other members of FOSC, follows the same mechanism of entry and colonization process in the xylem before the wilting of plants takes place. For the successful infection and colonization in the host plants, a complex and coordinated interplay of enzymes is required, including cell wall degrading enzymes, which probably act as key players in the pathogenesis and growth. A difference in the proteins was found among the data taken, i.e., presence of proteins in the treated compared to mock samples, especially the proteins belonging to hydrolase and oxidoreductase families and the activities of these enzymes had shed light on the mechanism of infection while inside the host plant as also reported in case of Fusarium graminearum (Paper et al. 2007). Further, the carbohydrate metabolism process was found signi cant with hydrolase and oxidoreductase activity, re ecting the importance of these CAZYmes during the pathogenesis.
Perhaps, most of the CAZymes annotated in the Fol proteins were described as hypothetical proteins (HP); therefore, the functional assignment to these HP was also achieved in the present study. Interestingly, the CAZymes identi ed, such as AA3 (Schmidt et al. 2013), belonged to gene accessions that were present on conditionally dispensable chromosomes (CDC), also known as supernumerary chromosomes (SC) or lineage-speci c (LS) chromosomes, though not on Fol CDC but GH3 and GH43 were reported to be present on legume infecting formae speciales CDC (Williams et al. 2016). These chromosomes are known to determine the pathogenicity in Fusarium oxysporum formae speciales (Ma et al. 2010;Vlaardingerbroek et al. 2016;Williams et al. 2016;van Dam et al. 2017). At the same time, the majority of CAZymes were predicted to be soluble and extracellular proteins involved in the degradation of polysaccharides, either of plant or fungal origin. However, two CAZymes families: GH47 (alpha-mannosidase) and GH72 (β-1,3-glucanosyl transglycosylase), were membrane proteins harboring signal peptides.
Among these non-secretory CAZymes, GH72 (β-1,3-glucanosyl transglycosylase), a membrane protein, was revealed to help in fungal cell wall formation and attachment of glycoprotein into the cell wall (Ao and Free 2017;Kar et al. 2019). Likewise, another membrane protein, GH47 (α-mannosidase) was also reported to be involved in cell wall morphogenesis with a secretory CAZyme GH20 (β-hexosaminidase) (Wymelenberg et al. 2010). However, not being extracellular but the presence of these CAZymes in the xylem sap suggest them to be important in fungal growth and survival while inside the plant.
The secretory CAZymes reported to be involved in fungal cell wall degradation, GH55 (β-1,3-glucanase) seemed to have a role in cell wall morphogenesis and germination (Millet et al. 2019). Moreover, an important family and much explored CAZyme, GH18 (Chitinase), identi ed in the present study signi ed its role in morphogenesis, defence, or parasitism (Oyeleye and Normi 2018) because of its involvement in the breakdown of chitin, an essential component of the fungal cell wall for cell growth and plasticity (Langner et al. 2015). Further, the presence of these two CAZymes viz. GH55 and GH18 were also reported to be found in the secretome of Aspergillus niger (Nitsche et al. 2012). Meanwhile, the GH families, especially GH20 (β-hexosaminidase), was reported to be involved in the trans-glycosylation process (Muschiol et al. 2020) and helps in fungal hyphal growth and branching (Rast et al. 1991). Additionally, the role of this family had also been shown to assist the chitinase for the complete degradation of chitin (López-Mondéjar et al. 2009). Moreover, the family GH32 (invertase) involved in the hydrolysis of sucrose reported here, perhaps, indicated its signi cant role in an increased level of pathogenicity (Ruiz and Ruffner 2002;Voegele et al. 2006). However, its actual role in plant-pathogen interaction remains unclear (Chang et al. 2017).
Further, the rest of the extracellular CAZymes, including AA3, an aryl-alcohol or glucose oxidases, was identi ed to play a role in biomass degradation as in the case of a necrotrophic fungus, Botrytis cinerea (Petrasch et al. 2019). Likewise, AA5 (a galactose, ra nose or alcohol oxidase) that oxidize oligogalacturonides simultaneously was reported to reduce the activation of plant immune response (Ferrari et al. 2013).
Pectinolytic enzymes are very crucial in the process of pathogenesis because of their role in plant cell wall degradation and the CAZyme, CE8 (pectin methylesterase), the only CE family identi ed with GH28 (polygalacturonase) was identi ed to be involved in pectin degradation by a de-esteri cation process, which in turn, allows the pectin accessible to other pectinases such as polygalacturonase (Pelloux et al. 2007), while GH28 was reported to be engaged in the breaking of α-linked galacturonic acid bonds (Markovič and Janeček 2001) in the pectin. Moreover, the presence of these two CAZymes families was also reported to be co-expressed in a basidiomycetes fungus (Miyauchi et al. 2016) and identi ed in the secretome of an ascomycetes species (Couturier 2016).
To get an insight into the coordination of these CAZymes, an interaction network of the CAZymes based on the evidence from various sources resulted in the identi cation of six CAZymes, i.e., GH3, GH18, GH20, GH28, GH43 and CE8 to be directly involved in the interactions out of which excluding GH3, the other CAZymes were enriched for a metabolic pathway involved in pectin and chitin degradation. Although enriched for and belonging to different cell wall degrading categories, i.e., cellulose/hemicellulose, hemicellulose and pectin, their coordination might be playing a signi cant role in fungal growth and pathogenesis. Besides this, GH3 also possessing chitinolytic activity and interacting with GH18, may involve in the chitin degrading mechanism (Yang et al. 2014), suggesting their signi cant role in the process of morphogenesis of the fungus. Meanwhile, their interconnected network, along with the contribution of CE8, which also assist other polygalacturonases, is found to be interacting with a pectate lyase and a xylosidase, where both of these families in a broad sense considered as plant cell wall degrading enzymes. Therefore, the coordination of these CAZymes in the network (including GH43, hemicellulose degrading CAZymes) and their presence in the xylem sap implicates a role in fungal colonization, most probably by degrading complex sugars into simpler sugar forms for growth and morphogenesis. Another CAZymes, i.e., GH55, although not directly reported to be involved in the main network, but was observed to be linked with a GH17 family, perhaps contributing to the phenomenon of cell morphogenesis and conidiation (Millet et al. 2018(Millet et al. , 2019, which is further connected to GH32 (invertase), an enzyme involves in breaking down of sucrose sugar abundantly found in plants.
Hence, within the intricate mechanism of cell wall degradation during the process of colonization by this fungus, some of the crucial CAZymes families, i.e., AA3, GH3, GH18, GH20, GH28, GH43, GH55 and CE8, were identi ed in the xylem sap of a susceptible host plant, which could be key players in the pathogenesis and also gave a glimpse on the mechanism of colonization among the interacting CAZymes through available evidence. Therefore, extracellular CAZymes identi ed in the xylem sap might have been involved in the growth, maintenance and colonization process of this pathogen.

Conclusion
Although several secretomes and transcriptomic studies in various pathogenic fungi were done, most of them were restricted to in-vitro set up only; hence an attempt was made to ful l this lacuna. Therefore, to identify and annotate the cell wall degrading CAZymes at the protein level and revealing the role of these CAZymes in the process of successful pathogenesis through cell wall degradation and also in fungal survival, the present study of in-planta proteomics analysis of two independent data sets was done to elucidate cell wall degrading CAZymes families of fungal origin. Some of the families belonging to AA, CE and GH were identi ed and their potential role in fungal colonization and pathogenicity as understood was discussed. Hence, this study provides a future scope for detailed studies on these candidate CAZymes within the formae speciales and to depict their elaborate mechanisms during pathogenesis.

Declarations Funding
No funding was received for conducting this study.

Con icts of interest/Competing interests:
The authors have no con icts of interest to declare that are relevant to the content of this article.

Availability of data and material
All data generated or analysed during this study are included in this published article (and its supplementary information les) Code availability NA Authors' contributions AR and PTV designed the study. AR BK and AJ performed the analysis. AR, BK, AA and PTV wrote the nal manuscript. All the authors analyzed and approved the manuscript. (* removal of contaminant, reverse hits, identi ed by site and peptide count <2.These proteins were belonging to majority protein groups after processing in each experiment having four replicates were identi ed by the peptides present in more than two replicates)