Dual transcriptome sequencing and mapping
To understand the saffron-Fusarium oxysporum R1 interaction mechanism, dual transcriptome of Fox R1 inoculated saffron corms were sequenced and compared with the mock inoculated corms at 2 days post inoculation (Fig. 1). The total number of raw reads obtained after RNA sequencing ranged from 55 to 85 million, 150bp paired end reads per sample. The mapping of raw reads to Fusarium oxysporum f.sp. lycopersici 4287 reference genome resulted in 0.22% and 2.81% alignment in mock inoculated and Fox R1 inoculated saffron corms respectively (Table S3a). The unmapped reads were of saffron corm, were de-novo assembled and resulted in 71161 contigs with minimum contig length of 501 and maximum contig length of 16008. The N50 and N80 value of the assembled genome was 1071 and 667 respectively (Table S3b).
Differentially Expressed Genes Of Fox R1 And Functional Annotation
A total of 7,876 significant DEGs of Fox R1 were identified in the transcriptomics data considering mock inoculated sample as control. Among them, 46 genes were found common in mock inoculated and Fox R1 inoculated samples. The common genes were less because control corms were not inoculated with Fox R1, so the common genes could be of the Fusarium oxysporum strain CSE15 endophyte present in both the samples (Wani et al. 2016). Out of 46 common, 39 genes were up-regulated and 7 were down-regulated. In Fox R1 inoculated samples 7830 genes were identified, of which 1049 significant genes were with predicted functions (Data not shown) and remaining genes were hypothetical. The 57 putative genes related to virulence and pathogenicity (such as FRP1, FOW1, FOW2, CHSV, FGA1, FGB1, FMK1, STE12, SGE1 etc.) was identified after annotated the data with PHI (Pathogen-Host interaction). The genes were identified considering Fusarium oxysporum as reference and around 68.3% of Fox R1 genes have shown 100% sequence similarity (Table S4). The results indicated the conservation of genes related to virulence in different f.sp. of Fusarium oxysporum.
Gene Ontology And Kegg Analysis
Further, the genes were subjected to GO analysis, 3318 GO terms were assigned of which 1111 (33.5%) GO terms were related to molecular function, 1759 (53%) biological process and 448 (13.5%) were related to cellular component. The 270 significantly enriched GO terms (sorted by p value < 0.05) were identified. The top 10 enriched GO terms in each category was plotted against number of genes (Fig. 2). The most enriched GO terms related to molecular function was binding (GO:0005488) and purine ribonucleoside triphosphate binding (GO:0035639), and in cellular component was protein-containing complex (GO:0032991) and intracellular anatomical structure (GO:0005622) and in biological process was cellular process (GO:0009987) and cellular metabolic process (GO:0044237). The genes were further classified in to 124 KEGG pathways, 18 KEGG pathways were found enriched based on significant p value (p < 0.05). The top 5 enriched pathways were related to Oxidative phosphorylation (fox00190), Spliceosome (fox03040), Protein processing in endoplasmic reticulum (fox04141), Autophagy (fox04138) and Endocytosis (fox04144) (Fig. 3). This suggests that during the infection process in Fox R1, the genes/pathways related to ATP and protein synthesis has been activated as during infection pathogens secrete different proteins involved in pathogenicity.
Pathogenicity and virulence genes of Fox R1
The present study focused only on the differentially expressed Fox R1 genes related to virulence and pathogenicity. The virulence and pathogenicity genes expressed by Fox R1 were plant cell wall degrading enzymes, transcription factors and various signaling molecules. All these virulence factors get activated after the initiation of signaling cascade. The signaling cascade gets activated in Fusarium oxysporum after the correct identification of the host. The identification of correct host is the prerequisite for the vascular colonization of the host plant (Husaini et al. 2018). The first players involved in the signaling cascade are the receptors that perceive the signal molecules from the host.
Membrane proteins and role in pathogenicity In Fusarium oxysporum, membrane proteins act as receptors and play a significant role in regulating the infection process. The two membrane proteins i.e. Msb2 and Sho1 contribute to pathogenicity by regulating the Fmk1 pathway (Perez-Nadales and Pietro, 2015) (Fig. 4B). Msb2 is a mucin type highly glycosylated membrane protein that work in cooperation with another membrane protein Sho1. Sho1 is a tetraspan transmembrane protein; both are required for the phosphorylation of fmk1 gene and regulate invasive growth and pathogenicity of Fusarium oxysporum (Perez-Nadales et al. 2014, Perez-Nadales and Di Pietro, 2015). In the present study gene encoding for Msb2 (FOXG_09254) and Sho1 (FOXG_06120) of Fox R1 were up-regulated 12.6 log2fold and 13.2 log2fold respectively during colonization of saffron corm.
Mitogen activated protein kinase (MAPK) signaling pathway The stimulation of signal activates specific signaling cascade in the cell. The mitogen activated protein kinase (MAPK) signaling pathway is one of the most conserved mechanisms that process the extracellular signals inside the cell and in turns activates different targets such as transcription factors (Martinez-Soto and Ruiz-Herrera, 2017). Fmk1 (Fusarium-mitogen kinase 1) is the important MAPK that regulate the invasive growth of Fusarium oxysporum and largely dependent on the transcription factor Ste12. Fmk1 also regulate the ability of Fusarium oxysporum to colonize the roots of the host plant and deletion of fmk1 gene resulted in impaired root attachment in Fusarium oxysporum (Perez-Nadales et al. 2014; Di Pietro et al. 2001). In addition, Fmk1 regulate the expression of genes i.e. chsV gene encoding chitin synthase V, fks1 encoding 1,3-beta-D-glucan synthase, gas1 encoding β-1,3-glucanosyltransferase involved in cell wall synthesis of Fox (Husaini et al. 2018) (Fig. 4E). In the present study, the expression of gene encoding Fmk1 (FOXG_08140) was up-regulated and it was 12.6 log2Fold. However, Guo et al. (2014) have reported the expression of fmk1 gene was not induced in Fusarium oxysporum f.sp. cubense race 1 and race 4 at 48h post inoculation in banana plant but the expression of TF Ste12 has increased in Fusarium oxysporum f.sp. cubense race 4 (Foc 4) indicating invasive growth pathway activation in Foc 4. fmk1 RNAi transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings (Pareek and Rajam, 2017). Further, RHO type GTPase Rho1 regulates the post transcriptional activity of glucan synthase and is required for the correct assembly of the cell wall (Martinez-Rocha et al. 2008). In the present study, rho1 gene (FOXG_00386) has shown an up-regulation of 12.1 log2fold.
Hog1 and Pbs2 are the two other MAP kinases involved in HOG (high osmolarity glycerol) pathway and have role in oxidative stress, hyperosmolarity response and glycerol synthesis. In the present study, both the genes hog1 and psb2 have shown up-regulation of 14.4 log2Fold and 11.7 log2Fold respectively. The expression of MAPK Hog1 is under the control of histidine kinase Fhk1 (Fig. 4C). Pareek and Rajam (2017) have reported the reduction in conidial size, reduction in invasive growth and significant decrease in pathogenesis of hog1 and psb2 RNAi transformants of Fusarium oxysporum on tomato seedlings compared to wild type strains.
G-proteins G-proteins present on the cytoplasmic surface of the cell membrane are the essential components that mediate cellular response to external signal in fungus (Husaini et al. 2018). When extracellular signals bind to the G-protein couple receptors (GPCR), the receptor undergoes confirmation and in turn actives trimeric G protein (α, β and ȣ) that binds GTP molecule. In Fusarium oxysporum, two α G proteins i.e. FGA1 and FGA2 and one β G protein i.e. FGB1 have been identified and characterized. In the present study, fga1 and fgb1 genes were identified in PHI database and both have shown up-regulation during colonization of Fox R1.
FGA1 regulates growth and development in Fusarium oxysporum. α subunit of G- proteins have also been reported to regulate the activity of adenylate cyclase and intracellular cAMP level that result in pathogenicity (Fig. 4D). Deletion of fga1 gene resulted in low level of intracellular cyclic AMP, reduction in condition and pathogenicity of Fusarium oxysporum (Jain et al. 2002). Guo et al. (2016) have reported that deletion of fga1 gene in Fusarium oxysporum f.sp. cubense resulted in phenotypic effects in vegetative growth, colony morphology and pathogenicity in banana plant. Jain et al. (2003) reported that the deletion of gene fgb1 resulted in colony morphology, conidia formation, germination frequency, and decrease in intracellular cAMP concentration and reduced pathogenicity. It was concluded that both FGA1 and FGB1 G-proteins have overlapping functions in Fusarium oxysporum.
Transcription factors (TFs) Transcription factors are the proteins that regulate the expression of genes involved in different pathways implicated in Fusarium oxysporum virulence (Husaini et al. 2018; John et al. 2021). Till date, about 700 TFs have been envisaged and only 26 have been functionally characterized in different f.sp. of Fusarium oxysporum (Zuriegat et al. 2021). Among 26 TFs, 23 have reported role in pathogenicity and belongs to 10 different families of transcription factors. In the present study, 15 TFs out of 23 having role in virulence has been identified in Fusarium oxysporum R1. 4 TFs (Ctf1, Fow2, XlnR, Ebr1) belong to transcription family (Zn(II)2Cys6). 3 TFs (Con7-1, ZafA, Ste12) belonging to C2H2 transcription family, 1 TF (MeaB) from bZIP family, 1 TF (Snt2) from PHD family, 1 TF (Fnr1) from GATA family, 2 TF (VeA, VelB) from Velvet family, TF (Sge1) from Gti1/Pac2 family, TF (Ren1) from HSF family (Table 1). These TFs work together in different combination to regulate particular pathway in Fusarium oxysporum. The transcription factor Sge1 (six gene expression 1) (FOXG_10510) regulates the expression of SIX (secreted in xylem) genes (Fig. 4G). The Sge1 TF was first identified in Fusarium oxysporum f.sp. lycopersici and the deletion of SGE1 gene in this pathogen resulted in the loss of virulence in tomato plant (Michielse and Rep, 2009). In Fusarium oxysporum f.sp. cubense tropical race 4 (Foc TR4) and race 1, Sge1 is required for virulence in banana roots (Fernandes et al. 2016). In the present study, during Fox R1 infection, 11.4 log2Fold expression of gene encoding TF Sge1 was recorded. However, the SIX genes were not expressed. The TF XlnR (FOXG_03748) is the transcriptional regulator of the xylanase gene that code for CWDE xylanase (Fig. 4H) (Garcia-Enciso et al. 2018; Zuriegat et al. 2021). It has been reported that in Fol, deletion of the xlnr gene resulted in deactivation of xylanase genes during infection of tomato plant. However, the xlnr mutants were still fully pathogenic on tomato plants (Calero-Nieto et al. 2007). These results revealed that TF XlnR, the key transcriptional activator of xylanase genes, is not an essential virulence determinant in Fusarium oxysporum (Calero-Nieto et al. 2007).
Table 1
List of different categories of virulence factors identified in Fox R1 during infection in saffron corm.
S. No. | Categories | Fox id | Gene name | Product | Log2fold change (T vs C) |
1 | Plant cell wall degrading enzymes | FOXG_19077 | pl1 | Pectin lyase | 13.2 |
2 | FOXG_12330 | pme | Pectinmethylestrase | 12.5 |
3 | FOXG_13051 | pg5 | Endopolygalacturonase | 11 |
4 | FOXG_08862 | pgx1 | Exopolygalacturonase | 10.7 |
5 | FOXG_15742 | xyl4 | Endo-beta-1,4-xylanase | 9.4 |
6 | Mycotoxin | FOXG_02322 | fub1 | Fusaric acid | 11.3 |
7 | Transcription factors | FOXG_03748 | xnlr | Zn(II)2Cys6 TF | 13.6 |
| FOXG_06378 | fow2 | Zn(II)2Cys6 TF | 13.2 |
| FOXG_04196 | ctf1 | Zn(II)2Cys6 TF | 10.1 |
| FOXG_05408 | ebr1 | Zn(II)2Cys6 TF | 9.9 |
| FOXG_02103 | ste12 | C2H2 TF | 13.5 |
| FOXG_11503 | con7-1 | C2H2 TF | 11.9 |
| FOXG_00370 | zafa | C2H2 TF | 11 |
| FOXG_02277 | meab | bZip TF | 12.5 |
| FOXG_03165 | fnr1 | GATA TF | 12.1 |
| FOXG_01993 | snt2 | PHD TF | 11.3 |
| FOXG_10510 | sge1 | Gti1/Pac2 family | 11.4 |
| FOXG_10430 | ren1 | HSF TF | 9.3 |
| FOXG_09390 | ftf2 | Fusarium-transcription factor2 | 15 |
| FOXG_11273 | vea | Velvet family | 8.18 |
| FOXG_00016 | velb | 12.5 |
| Mitogen activated protein kinase | FOXG_08140 | fmk1 | Fusarium-mitogen kinase 1 | 12.6 |
| FOXG_06318 | hog1 | | 14.4 |
| FOXG_03107 | pbs2 | | 11.7 |
| G-proteins | FOXG_09359 | fga1 | Guanine nucleotide-binding protein alpha subunit | 11.9 |
| FOXG_11532 | fgb1 | Guanine nucleotide-binding protein beta subunit | 13 |
| Membrane proteins | FOXG_09254 | msb2 | Mucin rich membrane protein | 12.6 |
| FOXG_06120 | sho1 | Tetraspan membrane protein | 13.2 |
| FOXG_00386 | rho1 | Monomeric G-protein | 12.1 |
Velvet complex is heterotetrameric complex consists of four components i.e. VeA (FOXG_11273), VelB (FOXG_00016), VelC (FOXG_02050) and LaeA (FOXG_00975) (Fig. 4I) that is required for fungal development and secondary metabolite formation (Wang et al. 2022). In Fol, the VeA and LaeA are required for full virulence and deletion of gene VEA, VELB resulted in increased condition but changes in the shape and size of microconidia (Lopez-Berges et al. 2013). In the present study, all the components of velvet complex have been expressed and their expression was 8.18 log2Fold, 12.5 log2Fold, 10.4 log2Fold and 11.7 log2Fold respectively in Fox R1.
Transcription factor Ste12 (FOXG_02103) regulates the expression of genes involved in pathogenicity and act downstream in MAPK pathway (Rispail and Di Pietro, 2009). In Fol, mutants lacking Ste12 gene showed dramatic defects in secretion of pectinolytic enzymes, invasive growth, vegetative hyphal fusion, and as well as reduced virulence in tomato plants (Rispail and Di Pietro, 2009). Ste12 also positively regulates extracellular amylase and cellulose activities in Fol (Rispail and Di Pietro, 2009). In the present study, gene encoding TF Ste12 up-regulated 13.5 log2Fold in Fox R1 during invasion. In addition, the three TFs i.e. Ebr1, Con7-1 and Snt2 have been reported to affect general metabolism in Fusarium.
Ebr1 (Enhanced branching 1) is a TF belongs to Zn(II)2Cys6 family and is encoded by the gene present on the pathogenicity chromosome in Fusarium oxysporum (Van der does et al. 2016; John et al. 2021). Deletion of ebr1 gene in different Fol strains containing different numbers of ebr genes resulted in similar impaired growth and reduced pathogenicity, suggesting that loss of ebr1 causes a growth and pathogenicity phenotype in Fol strains independent of the other ebr copies (Jonker et al. 2014). However, in the present study, only one gene encoding Ebr1 (FOXG_05408) was identified in Fox R1. Con7-1 TF is the most important regulator of morphogenesis and virulence in Fusarium oxysporum (Ruiz-Roldan et al. 2015). In Fusarium oxysporum, three genes are present that encode for TF Con7-1 and two copies of Con7-2. These TF shows significant similarity with Con7p of rice pathogen Magnaporthe oryzae. It has been reported that Con7-1 in Fox regulates the expression of large number of genes involved in various biological processes such as morphogenesis and development, interaction with host plant, transduction, synthesis of primary and secondary metabolites, and regulation of genes encoding proteins such as polygalacturonases, cellulases and Six proteins (Fig. 4F, G) (Zuriegat et al. 2021). Deletion of gene encoding for Con7-1 in Fox resulted in impaired growth and reduced pathogenicity in tomato plants. However, deletion of gene encoding TF Con7-2 in Fox has not affected the pathogenicity (Ruiz-Roldan et al. 2015). In the present study, gene ecoding Con7-1 (FOXG_11503) was up-regulated 11.9 log2Folds. However, gene encoding Con7-2 was not expressed in Fox R1. Snt2 TF is well studied transcription factor in Fusarium oxysporum f.sp. melonis (Fom). It belongs to PHD transcription factor family. In Fom, this TF is involved in regulation of growth parameters such as hyphae growth & septation, conidiation. Targeted deletion of snt2 gene resulted in lower colonization of Fom to muskmelon stem and reduced virulence. This suggested that the TF Snt2 is required for pathogenesis in the early stages of infection process (Denisov et al. 2011). In the present study, all the three TF (Ebr1, Con7-1 and Snt2) have shown up-regulation in Fox R1 during the colonization process.
Fow2 is a transcription factor that is highly conserved in different forma specialis of Fusarium oxysporum (Husaini et al. 2018). One copy of gene encoding Fow2 is located on the core genome of Fox strain (chromosome number 6) whereas two paralogs are located on the lineage specific chromosome (Nino-Sanchez et al. 2016). In Fom, deletion of fow2 gene resulted in complete loss of pathogenicity (Imaxaki et al. 2007) and in Fol, deletion mutation resulted in 50% reduction in virulence compared to wild type (Michielse et al. 2009). In the present study, only one copy of gene (FOXG_06378) located on the core genome has been expressed.
Plant cell wall degrading enzymes Pathogenic fungi are known to secrete various types of extracellular enzymes that help them to invade the host tissue (Sharafaddin et al. 2019). During invasion, plant cell wall becomes the first barrier for the pathogen’s endeavor to colonize the plant tissue (Bacete et al. 2017). In response to this barrier, pathogens secrete specific extracellular enzymes known as cell wall degrading enzymes (CWDEs) and are considered as pathogenicity factors (Paccanaro et al. 2017). Furthermore, the CWDEs profile has also been used to distinguish different species of the Fusarium. The high virulent strains of Fusarium oxysporum causing Fusarium Yellow disease of chickpea have been reported to show different pectinase activity pattern compared to less virulent strains (Zamani et al. 2001; Motallebi et al. 2002).
In the present study, the expression of genes encoding for five different CWDEs i.e. pectin lyase (FOXG_19077), pectinmethylestrase (FOXG_12330), exopolygalacturonase (FOXG_08862), endo-beta-1,4-xylanase (FOXG_15742), and endopolygalacturonase (FOXG_13051), were induced during the colonization of Fox R1 inside the corm tissue (Table 1). All these enzymes play an important role in virulence as there is a correlation of CWDEs and the pathogenecity of fungi. Polygalacturonase are the type of pectinases that are secreted by pathogenic fungi and specifically act on the pectin, a component of cell wall of plants (Lorrai and Ferrari, 2021). The CWDE exo-polygalacturonase pgc4 has been reported as virulence factor in Fusarium oxysporum f.sp. cubense race 4, a pathogen of banana (Dong et al. 2020b). Recently, Zhu et al. (2021) have reported the exopolygalacturonase-related genes as susceptible factor in Potato-Verticillium dahlia interaction. The endo-beta-1,4-xylanase are the type of xylanase enzymes that specifically break the bond β (1–4) bonds present in the xylan and produce xylooligosaccharides as end product. The genes encoding this enzyme have been reported as virulence factors in various phytopathogens such as Fusarium oxysporum (Gomez-Gomez, 2002), Botrytis cinerea (Noda et al. 2010), Sclerotinia sclerotiorum (Yu et al. 2016), Valsa mali (Yu et al. 2018), Verticillium dahlia (Wang et al. 2021). The pectinolytic enzymes pectin lyase and pectin methylesterase (PME) are the first degrading enzymes to be induced and expressed in pathogenic fungi during the colonization process (Lu et al. 2019). PME causes desertification of pectin by removing the methoxy ester groups after desertification of pectin, the other cell wall degrading enzymes can easily act on cell wall components (Sella et al. 2016). Pectin lyase causes depolymerization of pectin and acts on the internal glycosidic bonds. Among the different genes encoding CWDE, the highest expression was shown by pectin lyase and pectin methylesterase in the present study (Table 1).
For the validation of RNA seq data, 20 genes were randomly selected for quantitative Real time PCR.The expression of all the genes have been quantified and compared with the in-vitro grown Fox R1 (Fig. S1). All the gens of Fox R1 expressed in-planta have shown up-regulation compared to in-vitro growm Fox R1. Though, the Fox R1 is different than the Fusarium oxysporum f.sp. gladioli reported earlier as a pathogen of saffron but it seems to share similar genes and expression pattern as reported in f.sp. of Fusarium oxysporum that are plant pathogens such as F. oxysporum f. sp lycopersici, F. oxysporum f. sp. melonis, F. oxysporum f. sp. ciceri. The genes that were up-regulated on infection in various Fox strains were up-regulated here as well and should have some role in virulence and colonization. Some genes such as SIX genes were up-regulated in other strains were not affected/detected in present study, suggesting their limited role in the infection by Fox R1 which needs further validation.
De-novo Data Analysis, Differential Expression And Functional Annotation
A total of 10,128 differentially expressed (DEGs) significant genes (p value < 0.05) of corm were expressed in (T vs C) (Fig. 5a) of which 5212 genes (51.46%) were up-regulated and 4916 genes (48.54%) were down-regulated (Fig. 5b). For the functional analysis of the genes, these genes were subjected to BLAST against different databases and resulted in 6716 genes (66.3%) annotation with SWISS-PROT database, 6773 genes (66.8%) annotation with GO database, 5209 genes (51.4%) with pfam database, 106 genes (1.04%) were annotated with eggnog database and 734 genes (7.2%) by KEGG database (Fig. 5c). The functional annotation of the DEGs, were performed using four different databases; GO (Gene Ontology), COG (Cluster of Orthologous groups of Proteins), KEGG (Kyoto Encyclopedia of Genes and Genomes) and MapMan database.
Gene Ontology, Cog And Kegg Pathway And Mapman Analysis
GO and COG analysis A total of 6673 genes annotated with GO database were assigned GO terms. 33.8% GO terms were associated with Molecular Function (MF), 33.4% GO terms were associated with Cellular Components (CC) and 32.7% with Biological Process (BP) (Fig. 5d). The enriched relevant GO terms in response to fungal infection were response to stress (GO:0006950), defense response (GO:0006952), response to chitin (GO:0010200), response to wounding (GO:0009611), response to biotic stimulus (GO:0009607), regulation of jasmonic acid mediated signaling pathway (GO:2000022), response to fungus (GO:0009620), plant-type cell wall (GO:0009505), chitinase activity (GO:0004568) suggesting that DEGs involved in defense of saffron in response to Fox R1 infection has activated (Fig. 6). Similar to present study, Silvia Sebstiani et al. (2017) have reported that GO terms such as response to chitin, plant-type cell wall, response to fungus and chitinase activity as factor of resistance in banana to Fusarium oxysporum f. sp. melonis Snyd. & Hans race 1.2 (FOM1.2). In another study, the GO enrichment analysis revealed the GO term such as cellular process, binding, metabolic process, membrane, extracellular region, response to stimulus as enriched GO terms in soyabean in response to Fusarium oxysporum f.sp. batatas infection (Lin et al. 2017). It has been reported that over expression of particular “metabolic process” in BP category and other defense related categories during pathogenic Fusarium oxysporum strain 40 and soyabean interaction resulted in greater stimulation of soyabean defense (Lanubile et al. 2015). Further, the DEGs were annotated to COG database and assigned COG categories (Fig. 5e). Out of 26 major categories present in the COG database, 16 categories were found in the (T vs C) comparison. Among the different categories, the top three clusters based on the number of genes were ‘Translational, ribosomal structure and biogenesis; (14.1%) represented the largest cluster followed by ‘Amino acid, transport and metabolism’ (6.6%) and ‘General function prediction only (6.6%) (Fig. 5e).
Kegg Pathway Analysis
For the pathway analysis and enrichment, top 20 enriched KEGG terms were represented in bubble plot (Fig. 7). Among the 20 KEGG terms, top 5 enriched KEGG terms were ‘Metabolic pathway, Biosynthesis of secondary metabolites, Spliceosome, Plant hormone signal transduction’, MAPK signalling pathway–plant, indicated that the reprogramming of the host metabolism has occurred in response to Fox R1 attack (Fig. 7). The activation of MAPK signaling pathway, biosynthesis of secondary metabolites, Plant-pathogen interaction and production of plant hormone are known to mediate plant defense during pathogen attack (Tiwari et al. 2021). Similar to present study, Li et al. (2019) have reported the activation of pathway such as phenylpropanoid biosynthesis, biosynthesis of secondary metabolites and ribosome in banana in response to Fusarium oxysporum f.sp. cubense. Recently, Duan et al. (2022) have also reported the activation of pathways such as endocytosis, plant-pathogen interaction, MAPK signaling pathway plant, protein processing in endoplasmic reticulum and spliceosome in apple plant in response to Fusarium proliferatum f. sp. malus domestica strain MR5 infection. The genes involved in Plant-pathogen interaction (04626) pathways were studied in detailed in further sections.
Mapman Analysis
The significant genes of saffron corms in different comparisons were annotated to Mercator database of MapMan tool in order to elucidate the defense process in response to Fox R1 infection. The overview of the process that has been activated in different comparisons has shown in Fig. 8. The results indicated that the defense response in saffron occurred primarily through the activation of pathways such as hormone signaling, transcription factors redox state, PR- proteins, secondary metabolites, cell wall modification, MAPK pathway etc. The pathways related to plant hormone signaling, transcription factor families, PR-proteins, genes responsible for plant cell wall modification and redox state have major role to play in defense (Duan et al. 2022). So, DEGs related to these pathways were studied in detailed.
Signaling During The Saffron–fox R1 Interaction (Plant-pathogen Interaction)
During plant-pathogen interaction, the foremost step in the activation of plant defense is the successful recognition of the pathogen. Plants recognize pathogens by two different approaches: (a) the Pattern recognition receptors (PRPs) present on the cell surface of the plant cell that recognizes the pathogen-associated molecular patterns (PAMPs) on the surface of the pathogen to initiate the process of PAMP-triggered immunity (PTI) (b) intracellular receptors in the plant cell recognize the effector proteins secreted by the pathogen to initiate the process of effector triggered immunity (ETI) (Yuan et al. 2021; Chang et al. 2021). In plants, most of the receptors belong to the class of receptor like kinases (RLKs) and receptor like proteins (RLPs). These RKLS and RLPs based on extracellular domain are further divided into several sub families such as epidermal growth factor-like (EGF) domains, lysine motif (LysM) and leucine- rich repeat (LRR) (Mubassir, 2019). Among them, (LRR-RLKs) is the largest family in plants and plays important role in developmental and defense related pathways (Restrepo-Montoya et al. 2020). In the present study, 9 DEGs belonging to different classes of LRR-RLKs were identified such as LRR serine threonine protein kinase (STPK), different types of Brassinosteroid LRR receptor kinase such as (BRL2) and leucine rich repeat receptor BAK1 in Fox R1 infected corm tissue. Out of 9 DEGs, 6 were found to be up-regulated and 3 were down-regulated, their log2fold value has been tabulated in table 4.16. Among all the DEGs the highest expression was shown by receptor like kinase BAK1 gene i.e. 8 log2fold (Table S5). Brassinosteroid insensitive 1-associated receptor kinase (BAK1) is an important pattern recognition receptor required for PTI triggered immunity (Zhang et al. 2020). In previous reports, BAK1 have been reported to play role in resistance in banana against Fusarium oxysporum f.sp. cubense (Zhang et al. 2019) and in soyabean against Fusarium oxysporum strain 36 (Lanubile et al. 2015).
Chitin perception and signaling in saffron-Fox R1 interaction
Another important receptor and co-receptor complex was identified in present study that was involved in the perception of chitin. Chitin olignomers were percieved by plasma membrane receptor complex, lysine motif receptor like kinases (LysM-RLK) or lysine motif receptor like proteins (LysM-RLP) such as (CEBip) (Gong et al. 2020). In the present study, genes encoding (LysM-RLK) was not expressed. However, one gene encoding chitin elicitor-binding protein, a LysM-RLP was up-regulated 2.8 log2fold (Table S5). In Rice, OsCEBip has been identified as the high affinity receptor for chitin oligomer (Hayafune et al. 2014). Upon chitin perception, CEBip heterdimerize with another RLK i.e. CERK1 (Chitin elicitor receptor kinase 1) that act as co-receptor. Heterodimerization of receptor and co-receptor eventually leads to homodimerization of co-receptor. CERK1 plays a crucial role in intracellular chitin signaling (Gong et al. 2020). In the present study, one gene for CERK1 was up-regulated (1.9 log2fold). The up-regulation of both CEBip and CERK1 suggested the activation of PTI in saffron in response to Fox R1. Chitin activated CERK1 further activates downstream receptor like cytoplasm kinase (RCLK). In Arabidopsis, it has been reported that activated CERK1 further activates and phosphorylate PBL 19 and PBL 27 (serine threonine kinases), member of RCLK VII subfamily (Attia et al. 2020). Chitin activated PBL 19 and PBL 27 has been reported to activate MAPK signaling cascade and ROS burst (Couto and Zipfel, 2016). In the present study, genes encoding PBL 19 and PBL 27 were found to be up-regulated the expression of PBL 19 was 3.3 log2fold and of PBL 27 was 0.74 log2fold (Table S5). It is to be noted that saffron plant, for chitin perception follows the same pattern as in case of rice and for intracellular chitin mediated signaling it follows the same pattern as in case of Arabidopsis. So, elucidation of exact mechanism of chitin perception and intracellular signaling in saffron needs further investigation.
Ca2+ influx and signaling, role in plant defense
Ca2+ are recognized as secondary messenger and plays important role in plant immunity and defense (Boudsocq and Sheen, 2013). Intracellular variation in the calcium ion concentration is regarded as one of the early event after perception of environmental change (Aldon et al. 2018). It has been reported that chitin activated RCLKs phosphorylate and activate calcium nucleotide gated channels (CNGCs) to regulate chitin induced Ca2+ influx (He et al. 2019). CNGCs plays an important role in plants pathogen signaling by facilitating the Ca2+ uptake in to the cytoplasm (Ma, 2011; K Jha et al. 2016). In the present study, two genes encoding calcium nucleotide gated channels i.e. CNGC4 and CNGC8 were identified and both were found to be down-regulated (-1.5 log2fold and − 1.9 log2 fold) respectively suggesting their limiting role in saffron-Fox R1 interaction (Table S5).
Further, the Ca2+ message inside the cell is decoded by calcium binding proteins called calcium sensors. Plant calcium sensors are classified into three major groups: Calcium dependent protein kinases (CDPK), the calmodulin group (CaM) and Calmoudlin like protein (CML) family (Aldon et al. 2018). The activated CDPKs and (CaM/CML) further trigger activation of downstream signaling such as phosphorylation of respiratory burst oxidase homolog (Rboh) for ROS production (Gao et al. 2014) (Fig. 9). In the present study, four genes encoding CDPKs were up-regulated. Ashraf et al. (2018) have identified several components of calcium signaling in both resistant and susceptible cultivators of chickpea during Fusarium oxysporum f.sp. ciceri infection. In banana, MaCDPK1 and CDPK2 have been reported during Foc TR4 infection suggesting their role in biotic stress (Wang et al. 2016). Zhang et al. (2021) have identified CDPK in the vascular sap of banana infected with Fusarium oxysporum f.sp. cubense Tropical race 4.
The next key target of the CDPKs is Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), also called respiratory burst oxidase homologues (RBOHs) in plants. These are the critical enzymes that produce ROS (Reactive oxygen species) during the stress conditions. It has been reported that production of ROS is the hallmark of successful recognition of pathogen and induction of plant defense (Torres, 2010). Different functions of ROS has been postulated in response to pathogen such as activation of defense system of plants, regulation of production of phytoalexin and other secondary metabolites that arrest pathogen growth, but ROS are mainly associated with hyper sensitive response at the site of infection that causes programmed cell death and limit the spread of pathogen (Lam et al. 2001; Ali et al. 2018). In the present study, only one significant gene encoding RBOHA was found to be down-regulated suggesting limiting role of this gene in ROS production. Similar to present study, Guo et al. (2021) found that the endophytic strain Fo47 suppressed RBOHD expression to successfully colonize the root, indicating that RBOHD normally acts to restrict colonization. In Nicotiana benthamiana silencing of RBOHB gene resulted in reduced oxidative burst and reduced disease resistance against Phytopathora infestans indicating role of RBOHB in defense (Yoshioka et al. 2009). So, in the present study, it can can concluded that gene encoding RBOHA act as susceptible factor.
R- genes and ETI
Resistance (R) genes, the intracellular nucleotide-binding domain leucine rich repeat containing receptors (NLRs) are the most effective tool used by the plant to specifically recognize the effectors or proteins release by the pathogen, which activate plant immune response at the site of infection and constitute the second layer of immunity i.e. effector triggered immunity (ETI) (Yuan et al. 2021; Tena, 2021). R gene mediated recognition of effectors activate various defense signaling cascade and production of PR- proteins, which further activates the systemic acquired resistance in plants (SAR). SAR provides long term and broad spectrum resistance in plants against pathogens (Liu et al. 2007). In the present study, many R genes have been identified four of them have been shown by KEGG analysis also such as RIN4, PBS1, SGT1 and RAR1 (Fig. 9). The three genes (RIN4, PBS1, SGT1) were up-regulated and one gene i.e. RAR1 was found to down-regulated (-2.7 log2fold) (Table S5). In addition, two genes i.e. LRK10 and LRL21 were induced in presence in Fox R1. RIN4 (RPM1-interacting protein 4) is a small protein that plays role in regulating PTI and ETI. It has been reported that RIN4 is a negative regulator of PTI. The over expression of gene inhibits the PTI and absence of gene enhance the PAMP induced response in plants (Ray et al. 2019). In the present study, one gene for RIN4 was expressed and found to be up-regulated (1.8 log2fold).
Further, PBS1 is a resistant gene encoding a serine-threonine kinase that together with RPS5 gene recognize avrPphb effector released by the Pseudomonas syringae and provide resistance by hyper sensitive response (Swiderski and Innes, 2001). In the present study, one gene encoding PSB1 was found to up-regulated. Resistance to Pseudomonas syringae 2 (RPS2) is a leucine rich repeat resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express a virulence gene avrRpt2. In the present study, RPS2 gene was found to be down-regulated in presence of Fox R1.
RAR1 is a resistance gene first identified in barley that recognize effectors and provide resistance against powdery mildew disease (Shirasu et al. 1999). In rice RAR1 gene provide resistance against blast fungus Magnaporthe grisea (Thao et al. 2007). In the present study, RAR1 gene was found down-regulated in presence of Fox R1. LR10 gene of wheat encodes a resistance kinase that provides resistance against fungus Puccinia recondite f.sp. tritici causing leaf rust of wheat. This gene has been reported to provide resistance against leaf rust in sixteen different cultivars of wheat (Blazkova et al. 2002). In the present study, the gene LR10 was up-regulated 10.2 log2fold by Fox R1. Pik-1 gene encodes coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) proteins, that provide resistance again rice blast fungus Magnaporthe oryzae (Zhai et al. 2011). In the present study, the expression of Pik-1 gene was up-regulated 4.5 log2fold indicated the involvement of R genes in saffron defense in response to Fox R1.
Mitogen Activated Protein Kinase (MAPK) Pathways
The signaling mediated by both PTI and ETI activates the common downstream signaling particularly mitogen activated protein kinase (MAPK) signaling cascade (Bi et al. 2018; Yuan et al. 2021). The MAPK cascade plays a diverse role in disease resistance against pathogens. MAPK are one of the largest groups of enzymes that phosphorylated appropriate targets to regulate various function in the cells such as activation of various plant hormones, activation of transcription factors etc. (Jagodzik et al. 2018; Zhang and Zhang, 2022).
After activation, MAPK Kinase Kinase (MAPKKK/MEKK1) phosphorylates MAPK Kinase (MAPKK) which in turn phosphorylates MAPK. The activated MAPK further mediates the activation of the downstream genes (Zhang et al. 2018). In the present study as depicted by KEGG analysis (Fig. 9). The first gene involved in MAPK signaling is MEKK1 that is found non-significant in presence of Fox R1. MEKK1 further phosphorylate MKK1/2 and MKK4/5. In the present study, gene encoding MKK1, MKK2, MKK4 were found to be up-regulated (Table 2).
Table 2
List of genes of saffron corm related to MAPK cascade
Category | Trinity id | Gene id | Gene description | A vs C |
MAPK | TRINITY_DN26929_c0_g1 | M3K1_ARATH | Mitogen-activated protein kinase kinase kinase 1; | NS |
TRINITY_DN36821_c0_g2 | M2K1_ORYSJ | Mitogen-activated protein kinase kinase 1; | 0.92 |
TRINITY_DN28760_c0_g1 | M2K2_ARATH | Mitogen-activated protein kinase kinase 2; | 3.06 |
TRINITY_DN33131_c0_g1 | M2K4_ARATH | Mitogen-activated protein kinase kinase 4; | 1.01 |
TRINITY_DN35194_c0_g1 | MPK3_ARATH | Mitogen-activated protein kinase 3 {ECO:0000303|PubMed:8282107}; | 2.02 |
TRINITY_DN37499_c0_g2 | MPK4_ARATH | Mitogen-activated protein kinase 4 {ECO:0000303|PubMed:8282107}; | NS |
TRINITY_DN21481_c1_g1 | MPK6_ORYSJ | Mitogen-activated protein kinase 6; | 4.01 |
Further, MKK1/2 and MKK4/5 phosphorylate MAPK3, MAPK4 and MAPK6 which in turn activates WRKY transcription factor. It has been reported by biochemical and GOF analysis that MKK4/5 work upstream of MAPK3/6 in Arabidopsis (Ren et al. 2002). In presence of Fox R1, MAPK3 and MAPK6 were found to be up-regulated and MAPK4 was found non-significant (Table 2). The activation of MAPK3/4/6 has been reported as hallmark for immune system activation in plants (Bi et al. 2018). Gao et al. (2008) have also reported that the cascade MEKK1, MKK1/MKK2 and MAPK4 regulate innate immunity in plants. In Arabidopsis, MAPK3/MAPK6 phosphorylate WRKY33 and both component i.e. MAPK signaling and WRKY33 are essential for accumulation of camalexin (a major phytoalexin) in response to pathogen attack (Mao et al. 2011). Sebastitin et al. (2017) have also reported the up-regulation of MKK4 and MKK5 in resistant variety of melon against Fusarium oxysporum f.sp. melonis. Our transcriptome data indicated that defense signaling in saffron is largely dependent on MKK4/MKK5-MAPK3/6 cascade.
Transcription factors (TFs)
Transcription factors play a central role in plants by controlling the expression of the genes involved in the cellular processes (Baillo et al. 2019). In the present study, for the identification of transcription factor belonging to different families, the DEGs were annotated against the Plant Transcription factor Database (Pachon et al. 2007). In total, 735 TFs belonging to 54 families were identified in the present study (Table S6). The top ten representative families were bHLH (basic/helix-loop-helix), NAC (NAM, ATAF, and CUC), ERF family, MYB-related family, C2H2 related family, CH3 family, bZip, FAR1, WRKY and MYB family (Table S6).
Transcription factors belonging to 5 different families (bHLH, WRKY, MYB, NAC, and ERF) have been reported to play role in plant immunity (Tsuda and Somssich, 2015). Interestingly, all these families were highly expressed TFs families in the present study as well. Among different families, WRKY and ERF family has gained much attention due to their diverse role in growth, development and defense of the plant (Wani et al. 2021). The subfamilies of the WRKY family have been reported to be induced by PAMP-triggered immunity, microbe-assisted molecular pattern triggered immunity, effector-triggered immunity (Wani et al. 2021). In the present study, 17 genes encoding different WRKYs were induced and WRKY24 was the most up-regulated (9.29 log2fold) factor followed by WRKY72A, WRKY75, WRKY71, WRKY33, WRKY28 etc (Table S7). In the present study, the highest up-regulation was shown by WRKY24. WRKY24 have been reported to up-regulated in sugarcane leaves infected with Puccinia kuehnii during the early stages and expression was increased as the disease progress (Correr et al. 2020). The WRKY 75 and WRKY 28 have been reported to involve in SA, JA/ET signaling pathways and enhance resistance of the plant to fungal pathogens through the activation of these pathways (Chen et al. 2013). In tomato, WRKY33 have been reported to up-regulated during the infection caused by Fusarium oxysporum f.sp. lycopersici (Aamir et al. 2018). WRKY57 have been reported to be susceptible factor as it competes with WRKY33 and in turns affects the JA mediated signaling pathway (Birkenbihl et al. 2017). In the present study, down-regulation of WRKY57 during Fox R1 indicated that it acts as resistant factor in saffron-Fox R1 interaction however, it needs further validation.
Further, the genes encoding ERFs were up-regulated in response to Fox R1 but gene encoding AP2 subfamily of APR/ERF superfamily was down-regulated (Table S7). The down-regulation of AP2 subfamily in the present study indicated that this subfamily act as susceptible factor. AP2/ERF has been reported to induce resistance in Nicotiana benthamiana against Phytophthora parasitica (Yu et al. 2020), wheat against Puccinia striiformis f.sp. tritici (Hawku et al. 2021). Over expression of ERF1 & 2 genes in Arabidopsis, ERF1 in wheat & tobacco, ERF 3 in soya bean, ERF37, ERF49, ERF78 in wheat confers resistance to disease (Berrocal-Lobo and Molina, 2004; Chen et al. 2008, Zhang et al. 2009; Li et al. 2021) respectively. In the present study, genes encoding ERF105 up-regulated 10.8 log2fold followed by ERF17 (9 log2fold), ERF2 (3.5 log2 fold), ERF1 (1.6 log2fold). The factors factors ERF105 in maize, and ERF17 in beans have been reported to induce resistance against the pathogens (Zang et al. 2020; Guerrero-Gonzalez et al. 2011) respectively. ERF1 has been reported as the one of the marker involved in defense against fungal pathogens (Galindo-González and Deyholos, 2016).
Plant Hormones
Phytohormones have been reported to induce the defense response in plant against both biotic and abiotic stress. Fox R1 infection in saffron corm resulted in shift in the expression of genes encoding different hormones (Table S8). In the present study, 5 DEGs related to auxin biosynthesis and signaling were significantly up-regulated and 5 DEGs were down-regulated. Auxin is an important hormone that plays role in normal growth & development and also in plant-microbe interaction but the most studied class of auxin i.e. IAA (indole acetic acid) act as susceptible factor during the biotic stress (Kunkel and Johnson 2021) and also suppresses the stress the production of hormone salicylic acid (Galindo-Gonzalez and Deyholos, 2016). In the present study, the up-regulation of the auxin responsive protein IAA1, IAA3 and IAA16 suggesting their role as susceptible factor in saffron-Fusarium oxysporum interaction.
Further, 6 genes encoding two different enzymes i.e. isoforms of enzyme 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase involved in ethylene production up-regulated. In addition, the DEGs encoding the enzymes phenylalanine ammonia lyase (PAL) and 4-coumarate–CoA ligase-like-1, involved in the biosynthesis of salicylic acid was found up-regulated up-regulated more production of salicylic acid. Similar to present study, Lanubile et al. (2015) have also reported the induction of genes encoding for these two hormones during Fusarium oxysporum infection in soyabean plant. Jamail et al. (2019) have reported the more expression of PAL gene in rice plant treated with both bocontrol and pathogen in comparison to pathogen only. 1 DEG encoding jasmonic acid carboxyl methyltransferase 2 was up-regulated 4.83 log2fold. Jasmonic acid carboxyl methyltransferase 2 (JMT) is the key enzyme involved in the formation of methy jasmonate (ester of jasmonic acid); an important volatile plant hormone that regulate various processes in plants such as developmental processes, cellular processes and defense responses (Feng et al. 2021; Zhou et al. 2022). Jasmonic acid (JA) plays an important role in plant defense against both necrotrophic and hemibiotrophic fungus (Yang et al. 2019). The level of JA has been found critical for susceptibility or resistance in plants such as Arabidopsis, tomato, sweet potato against Fusarium oxysporum (Cole et al. 2014; Krol et al. 2015; Zhang et al. 2020). Hormones ethylene (Et) and jasmonic acid (JA) act synergistic in the regulation of both stress and developmental responses (Ma et al. 2020). The signaling of transcription factor ERF family (ethylene responsive factor) is mainly induced by combined action of JA and Et pathways. Salicyclic acid (SA) is known as defense hormone of plant as it protects plants from various pathogens (bacteria, fungi, viruses etc.). There is a co-relation between the endogenous level of SA and resistance against both hemibiotrophic and biotrophic fungus (Fusarium oxysporum, Alternaria alternata, Magnaporthe grisea, Colletotrichum gloeosporides, Xanthomonas spp) (Koo et al. 2020). SA induces SAR (systemic acquire resistance) in plants and is associated with the accumulation of PR-proteins (Vilasinee et al. 2019).
Pathogenesis related proteins (PR-Proteins)
The PR- proteins (Pathogenesis related proteins) are encoded by PR-genes. The PR proteins are the part of the innate immunity of plant as it accumulates at the site of infection during the pathogen invasion and also in the neighbouring uninfected tissue of the plant to minimize pathogen load (Jain and Khurana, 2018; Ali et al. 2018). PR proteins are also the part of the systemic acquired resistance in the plant against infection (Jain and Khurana, 2018). These proteins act as toxic proteins for the invading pathogens. PR proteins are present in all organs of the plant specifically more in leaves and are widely distributed in plant kingdom (Ali et al. 2018). They are thermo stable, protease resistant, low molecular weight (6-43kDa) and remain soluble at low pH (Jain and Khurana, 2018). They are divided into two subgroups acidic proteins that are secreted to extracellular space and basic proteins that are transported through vacuoles. Structurally, the PR proteins are divided into 17 families (PR1-PR17) and functionally these are classified into chitinase, germin like protein, thaumatin like protein, glucanases, lipid transfer protein, BARWIN, peroxidases etc. (Arora et al. 2020; Sharma et al. 2021). The ERFs have been reported to regulate the expression of the PR genes (Huang et al. 2016). In the present study, out of 17, 10 families of PR proteins were found to be induced (Table 3). Interesting, all the DEGs encoding different families of the PR proteins were significantly up-regulated except for 3 DEGs encoding different peroxidases (PR-9) that were found to be down-regulated.
Table 3
List of putative families of PR-proteins identified in saffron in response to Fox R1 infection
S.No. | PR-protein family | Description |
1 | PR-1 | Pathogenesis related protein-1 |
2 | PR-2 | β-1,3-Glucanases |
3 | PR-3 | Chitinase |
4 | PR-4 | BARWIN |
5 | PR-5 | Thaumatin like |
6 | PR-6 | Proteinase inhibitor |
7 | PR-9 | Peroxidase |
8 | PR-11 | Chitinase type 1 |
9 | PR-12 | Defensin |
10 | PR-14 | Lipid transfer protein |
As PR proteins are known to over express during the pathogen attack in plants; this seems to be true in saffron also. The highly up-regulated PR protein was BARWIN (11.9 log2fold), followed by beta-1,3-glucanase (11.6 log2fold), thaumatin like protein (10.5 log2fold), pathogenesis related protein-1 (9.4 log2fold), chitinase (7.5 log2fold) etc. BARWIN is a PR-4 protein, PR-4 family reported to have anti-fungal activity (Dabravolski and Frenkel, 2021). BARWIN was first identified in barley (Svensson et al. 1992) and later on, homologs of BARWIN was identified in many plants such as maize (Bravo et al. 2003), rice (Zhu et al. 2006), apple (Bai et al. 2013) etc. Franco et al. (2019) have identified and characterized homologs of BARWIN i.e. SUGARWIN1 and SUGARWIN2 in sugarcane as defense proteins. Further, beta-1,3-glucanases (PR-2) and acidic chitinases (PR-3) are the important hydrolytic enzymes that act on β-1,3-glucan and chitin, the structural components of the fungus cell wall (Kebede and Kebede, 2021). These two hydrolytic enzymes have been reported to be induce together after pathogen attack in maize (Laimbais et al. 1998), potato (Cheong et al. 2000), wheat (Li et al. 2001) etc. This seems to be true in case of saffron as well. Thaumatin-like protein (PR-5) is involved in plant defense against fungal pathogens and most of these have been reported to posses anti-fungal activity (Liu et al. 2021). The PR-1 family is a sterol binding protein, the members of this family are known to express abundantly during the pathogen attack and are used as a marker for salicylic acid mediated resistance (Breen et al. 2017). Various transcriptomics studies have revealed that the PR genes are significantly induced by biotic stress makes them one of the most promising candidates for developing multiple stress tolerant crop varieties (Gupta et al. 2013; Ali et al. 2018). In garlic, induction of PR-1 PR-2, PR-4 and PR-5 families have been reported during Fusarium oxysporum infection (Anisimova et al. 2021).
Reactive oxygen burst detoxification system of plants
During the pathogen attack, the reactive oxygen burst occurs in response to pathogen invasion as a part of the plant innate immunity. The pathogen invasion induces host genes for the ROS production as defense strategy, as these ROS react with DNA, lipids and other biomolecules causes cell death that prevent further spread of the infection). However, if left unchecked, these ions causes severe damage to plant cell so in order to prevent, plants encode enzymes (peroxidases, superoxide dismutase and Glutathione S-transferases) for the detoxification of ROS (reactive oxygen species). In the present study, 8 genes encoding different peroxidases (PR-9) were up-regulated and 3 down-regulated. Similarly, 3 genes related to glutathione dependent detoxification system were up-regulated and 2 gene encoding different Glutathione S-transferases were down-regulated (-7.3 log2fold). In addition, 3 DEGs encoding superoxide dismutase was up-regulated (Table S9). Galindo-Gonzalez1 and Deyholos, (2016) have reported the up-regulation of genes encoding peroxidase, superoxide dismutase in flax plant during Fusarium oxysporum f.sp. lini infection. Similar activation of detoxification enzymes have been reported in melon-Fusarium oxysporum f.sp. melonis interaction (Silvia Sebasitini et al. 2017), in chickpea- Fusarium oxysporum f.sp. ciceri interaction (Upasani et al. 2016), rough lemon-Plenodomus tracheiphilus interaction (Russo et al. 2021).
Structural modifications of cell wall
In response to pathogen attack, the plant cell wall also undergoes dynamic structural and chemical changes (Gorshkov et al. 2021). The major component of plant cell wall pectin is secreted in highly methylesterified form (Ren et al. 2019). Pectin methylesterase (PME)/ pectin esterases are the enzymes involved in the de-methylesterification and after this process cell wall become functional (Haas et al. 2021). The activity of PME is highly regulated by pectin methylesterase inhibitors (PMEI). The over expression of PMEI have been reported to reduce the activity of PME resulted in resistance of plant to pathogens (Wormit and Usadel, 2018). Increased activity of PME has been reported as susceptible factor in banana-Fusarium oxysporum interaction (Ma et al. 2013). In the present study, 3 DEGs encoding PMEI was up-regulated and 6 DEGs encoding PME was up-regulated (Table S10). The up-regulation of PME genes in present study, suggesting their roles as susceptible factors in saffron-Fusarium ocysporum R1 interaction. In wheat, the overexpression of PMEI has been reported to increases the pectin methyl esterification process and resistance against fungal pathogens Fusarium graminearum and Bipolaris sorokiniana (Vopli et al. 2011).
Further, the validation of RNA seq data was done. 20 genes were randomly selected for quantitative Real time PCR. The similar expression trend has been obtained in both the cases suggesting the reliability of the RNA seq data (Fig. 10).