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Fusarium wilt is one of the most destructive and less controllable diseases in melon, which is usually caused by fusarium oxysporum . In this study, transcriptome sequencing and Yeast Two-Hybrid (Y2H) methods were used for quantification of differentially expressed genes (DEGs) involved in fusarium oxysporum (f. sp. melonis race 1) stress-induced mechanisms in contrasted melon varieties (M4-45 “susceptible” and MR-1 “resistant”). The interaction factors of Fom-2 resistance genes were also explored in response to the plant-pathogen infection mechanism. Transcriptomic analysis exhibited total 1,904 new genes; however, candidate DEGs analysis revealed a total of 144 specific genes (50 upregulated and 94 downregulated) for M4-45 variety and 104 specific genes (71 upregulated and 33 downregulated) for MR-1 variety, respectively. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway depicted some candidate DEGs, including Phenylalanine metabolism, phenylpropane biosynthesis, plants-pathogen interaction, and signal transduction of plant hormones, which were mainly involved in disease resistance metabolic pathways. The weighted gene co-expression network analysis (WGCNA) analysis revealed a strong correlation module and exhibited the disease resistance-related genes encoding course proteins, transcription factors, protein kinase, benzene propane biosynthesis path, plants-pathogen interaction pathway, and glutathione S-transferase. Meanwhile, the resistance-related specific genes expression was relatively abundant in MR-1 compared to the M4-45, and cell wall-associated receptor kinases ( MELO3C008452 and MELO3C008453 ), heat shock protein (Cucumis_melo_newGene_172), defensin-like protein (Cucumis_melo_newGene_5490), and disease resistance response protein ( MELO3C016325 ), activator response protein ( MELO3C021623 ), leucine-rich repeat receptor protein kinase ( MELO3C024412 ), lactyl glutathione ligase (Cucumis_melo_newGene_36), and unknown protein ( MELO3C007588 ) were persisted by exhibiting the upregulated expressions. At the transcription level, the interaction factors between the candidate genes in response to the fusarium oxysporum induced stress, and Y2H screening signified the main contribution of MYB transcription factors ( MELO3C009678 and MELO3C014597 ), BZIP ( MELO3C011839 and MELO3C019349 ), unknown proteins, and key enzymes in the ubiquitination process ( 4XM334FK014 ). The candidate genes were further verified in exogenously treated melon plants with f. oxysporum ( Fom-2 , Race 1), Abscisic acid (ABA), Methyl Jasmonite (MeJA), and Salicylic acid (SA), using the fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis. The overall expression results indicated that the SA signal pathway is involved in effective regulation of the Fom-2 gene activity.

Breeding for disease resistance has been one of the most important research objectives in melon for the last few decades. Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) is among the most threatening melon fungal diseases along the Mediterranean coast, affecting yield and quality. Since genetic resistance is one of the best sustainable strategies that can be used to control this pathogen, 27 Tunisian melon accessions collected from local farmers have been tested using phenotypic and molecular approaches to identify new sources of resistance to be used directly as cultivars in affected areas or as resistance donors in breeding programs. The phenotypic evaluations, using artificial inoculations, showed several resistant accessions to the pathogenic races of Fom. Additionally, molecular analysis revealed that 13 out of 27 accessions carried the resistance Fom-1 gene (7 in homozygous state and 6 in heterozygous state), confirming their resistance to races 0 and 2. Two of them were also identified as heterozygous for the Fom-2 gene, being resistant to races 0 and 1. Furthermore, two accessions with a high level of resistance to the most virulent race 1.2 have been also reported. This melon germplasm should be explored as a potential source of resistance genes in breeding programs to develop new resistant melon cultivars.

Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by pathogens are called ‘avirulence’ proteins. Their identification is important for being able to assess the usefulness and durability of resistance genes in agricultural settings. We have used genome sequencing of a set of strains of the melon wilt fungus Fusarium oxysporum f. sp. melonis (Fom), bioinformatics-based genome comparison and genetic transformation of the fungus to identify AVRFOM2, the gene that encodes the avirulence protein recognized by the melon Fom-2 gene. Both an unbiased and a candidate gene approach identified a single candidate for the AVRFOM2 gene. Genetic complementation of AVRFOM2 in three different race 2 isolates resulted in resistance of Fom-2-harbouring melon cultivars. AvrFom2 is a small, secreted protein with two cysteine residues and weak similarity to secreted proteins of other fungi. The identification of AVRFOM2 will not only be helpful to select melon cultivars to avoid melon Fusarium wilt, but also to monitor how quickly a Fom population can adapt to deployment of Fom-2-containing cultivars in the field.

Three single nucleotide polymorphism (SNP) sites in which amino acids had changed were detected by sequence analysis within the leucine-rich repeat (LRR) region of the Fom-2 gene. Cleaved amplified polymorphic sequence (CAPS) and allele-specific PCR (AS-PCR) methods were employed to explore the SNP validation linked to fusarium wilt resistance in the F₁ and F₂ generations simultaneously. Homozygous- and heterozygous-resistant genotypes and homozygous-susceptible genotype could be clearly distinguished using the CAPS method, and three detected SNP sites were observed to be linked to fusarium wilt resistance, with a segregation ratio of 1:2:1 in the F₂ generation. In addition, heterozygous-resistant and homozygous-susceptible genotypes could be clearly distinguished in the F₁ generation using the AS-PCR method, showing a 3:1 segregation in terms of resistant and susceptible genotypes in the F₂ generation. We therefore developed SNP-based functional markers (FMs) and identified some melon germplasm resistant to fusarium wilt by FM analysis within melon species. In conclusion, the SNP-based FMs originating from the SNP site of the Fom-2 LRR region were determined to be linked to fusarium wilt resistance and showed promise in the enhancement of breeding in melon.

The melon gene Fom-2, which confers resistance to Fusarium oxysporum f.sp. melonis (Fom) races 0 and 1, has been previously characterized by map-based cloning, and it encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRRs). Here, we used the primer Fom2-LRR1639 to clone and sequence a partial LRR region of the Fom-2 gene in 11 melon accessions resistant to Fusarium wilt from various geographic regions. Our work revealed that the structure of the partial LRR domain is highly conserved between eight of these resistant accessions and is similar to the resistant allele in the previously characterized PI-161375 line. Conversely, PI-124111 is a unique line that presents the same resistant allele that was previously described in the MR-1 line. The accession Cum-355 presents a protein that differs from that encoded by both the resistant lines PI-161375 and MR-1. This result suggests that Cum-355 has a new resistant allele of Fom-2 that determines the same specificity. Importantly, based on the sequence of the Fom-2 LRR domain, two sequence characterized amplified region (SCAR) markers, Fom2-R408 and Fom2-S342, were developed for Fom-2 resistant and susceptible alleles, respectively. These allele-specific PCR markers could be used as co-dominant markers when their primer pairs were combined in a multiplex PCR reaction. The specificity of these functional markers (FM) was validated on a set of 27 genotypes representing several melon types. These FM markers are expected to enhance the reliability and cost-effectiveness of marker-assisted selection for the Fom-2 gene in melon.

Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and 1 of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that cosegregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.