When infecting a host plant, the fungus Fusarium oxysporum secretes several effector proteins into the xylem tissue to promote virulence. However, in a host plant with an innate immune system involving analogous resistance proteins, the fungus effector proteins may trigger resistance, rather than promoting virulence. Identity of the effector genes of Fusarium oxysporum f. sp. niveum (Fon) races that affect watermelon (Citrullus lanatus) are currently unknown. In this study, the SIX6 (secreted in xylem protein 6) gene was identified in Fon races 0 and 1 but not in the more virulent Fon race 2. Disrupting the FonSIX6 gene in Fon race 1 did not affect the sporulation or growth rate of the fungus but significantly enhanced Fon virulence in watermelon, suggesting that the mutant ΔFon1SIX6 protein allowed evasion of R protein-mediated host resistance. Complementation of the wild-type race 2 (which lacks FonSIX6) with FonSIX6 reduced its virulence. These results provide evidence supporting the hypothesis that FonSIX6 is an avirulence gene. The identification of FonSix6 as an avirulence factor may be a first step in understanding the mechanisms of Fon virulence and resistance in watermelon and further elucidating the role of Six6 in Fusarium-plant interactions.

Watermelon (Citrullus lanatus L.), which is an economically important cucurbit crop that is cultivated worldwide, is vulnerable to various adverse environmental conditions. Small heat shock protein 20s (HSP20s) are the most abundant plant HSPs and they play important roles in various biotic and abiotic stress responses. However, they have not been systematically investigated in watermelon. In this study, we identified 44 watermelon HSP20 genes and analyzed their gene structures, conserved domains, phylogenetic relationships, chromosomal distributions, and expression profiles. All of the watermelon HSP20 proteins have a conserved the α-crystallin (ACD) domain. Half of the ClHSP20s arose through gene duplication events. Plant HSP20s were grouped into 18 subfamiles and a new subfamily, nucleo-cytoplasmic XIII (CXIII), was identified in this study. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter regions of the watermelon HSP20 genes. Different from that in other species, half of the watermelon HSP20s were repressed by heat stress. Plant HSP20s displayed diverse responses to different virus infections and most of the ClHSP20s were generally repressed by Cucumber green mottle mosaic virus (CGMMV). Some ClHSP20s exhibited similar transcriptional responses to abscisic acid, melatonin, and CGMMV. Subcellular localization analyses of six selected HSP20- green fluorescence protein fusion proteins revealed diverse subcellular targeting. Some ClHSP20 proteins were affected by CGMMV, as reflected by changes in the size, number, and distribution of fluorescent granules. These systematic analyses provide a foundation for elucidating the physiological functions and biological roles of the watermelon HSP20 gene family.

Dwarf architecture is an important trait associated with plant yield, lodging resistance and labor cost. Here, we aimed to identify a gene causing dwarfism in watermelon. The ‘w106’ (dwarf) and ‘Charleston Gray’ (vine) were used as parents to construct F₁ and F₂ progeny. Dwarf architecture of ‘w106’ was mainly caused by longitudinal cell length reduction and was controlled by a single recessive gene. Whole-genome sequencing of two parents and two bulk DNAs of F₂ population localized this gene to a 2.63-Mb region on chromosome 9; this was further narrowed to a 541-kb region. Within this region, Cla015407, encoding a gibberellin 3β-hydroxylase (GA3ox), was the candidate gene. Cla015407 had a SNP mutation (G → A) in the splice acceptor site of the intron, leading to altered splicing event and generating two splicing isoforms in dwarf plants. One splicing isoform retained the intron sequences, while the other had a 13-bp deletion in the second exon of GA3ox transcript, both resulting in truncated proteins and loss of the functional Fe2OG dioxygenase domain in dwarf plants. RNA-Seq analysis indicated that expression of Cla015407 and other GA biosynthetic and metabolic genes were mostly up-regulated in the shoots of dwarf plants compared with vine plants in F₂ population. Measurement of endogenous GA levels indicated that bioactive GA₄ was significantly decreased in the shoots of dwarf plants. Moreover, the dwarf phenotype can be rescued by exogenous applications of GA₃ or GA₄₊₇, with the latter having a more distinct effect than the former. Subcellular localization analyses of GA3ox proteins from two parents revealed their subcellular targeting in nucleus and cytosol. Here, a GA3ox gene controlling dwarf architecture was identified, and loss function of GA3ox leads to GA₄ reduction and dwarfism phenotype in watermelon.

Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, which cause diseases in cucurbits, especially watermelon. In watermelon, symptoms develop on the whole plant, including leaves, stems, peduncles, and fruit. To better understand the molecular mechanisms of watermelon early responses to CGMMV infection, a comparative transcriptome analysis of 24 h CGMMV-infected and mock-inoculated watermelon leaves was performed. A total of 1641 differently expressed genes (DEGs) were identified, with 886 DEGs upregulated and 755 DEGs downregulated after CGMMV infection. A functional analysis indicated that the DEGs were involved in photosynthesis, plant–pathogen interactions, secondary metabolism, and plant hormone signal transduction. In addition, a few transcription factor families, including WRKY, MYB, HLH, bZIP and NAC, were responsive to the CGMMV-induced stress. To confirm the high-throughput sequencing results, 15 DEGs were validated by qRT-PCR analysis. The results provide insights into the identification of candidate genes or pathways involved in the responses of watermelon leaves to CGMMV infection.