The NF-Y Transcription Factor Family in Watermelon: Re-Characterization, Assembly of ClNF-Y Complexes, Hormone- and Pathogen-Inducible Expression and Putative Functions in Disease Resistance

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that binds to the CCAAT cis-element in the promoters of target genes and plays critical roles in plant growth, development, and stress responses. In the present study, we aimed to re-characterize the ClNF-Y family in watermelon, examine the assembly of ClNF-Y complexes, and explore their possible involvement in disease resistance. A total of 25 ClNF-Y genes (7 ClNF-YAs, 10 ClNF-YBs, and 8 ClNF-YCs) were identified in the watermelon genome. The ClNF-Y family was comprehensively characterized in terms of gene and protein structures, phylogenetic relationships, and evolution events. Different types of cis-elements responsible for plant growth and development, phytohormones, and/or stress responses were identified in the promoters of the ClNF-Y genes. ClNF-YAs and ClNF-YCs were mainly localized in the nucleus, while most of the ClNF-YBs were localized in the cytoplasm of cells. ClNF-YB5, -YB6, -YB7, -YB8, -YB9, and -YB10 interacted with ClNF-YC2, -YC3, -YC4, -YC5, -YC6, -YC7, and -YC8, while ClNF-YB1 and -YB3 interacted with ClNF-YC1. A total of 37 putative ClNF-Y complexes were identified, e.g., ClNF-YA1, -YA2, -YA3, and -YA7 assembled into 13, 8, 8, and 8 ClNF-Y complexes with different ClNF-YB/-YC heterodimers. Most of the ClNF-Y genes responded with distinct expression patterns to defense hormones such as salicylic acid, methyl jasmonate, abscisic acid, and ethylene precursor 1-aminocyclopropane-1-carboxylate, and to infection by the vascular infecting fungus Fusarium oxysporum f. sp. niveum. Overexpression of ClNF-YB1, -YB8, -YB9, ClNF-YC2, and -YC7 in transgenic Arabidopsis resulted in an earlier flowering phenotype. Overexpression of ClNF-YB8 in Arabidopsis led to enhanced resistance while overexpression of ClNF-YA2 and -YC2 resulted in decreased resistance against Botrytis cinerea. Similarly, overexpression of ClNF-YA3, -YB1, and -YC4 strengthened resistance while overexpression of ClNF-YA2 and -YB8 attenuated resistance against Pseudomonas syringae pv. tomato DC3000. The re-characterization of the ClNF-Y family provides a basis from which to investigate the biological functions of ClNF-Y genes in respect of growth, development, and stress response in watermelon, and the identification of the functions of some ClNF-Y genes in disease resistance enables further exploration of the molecular mechanism of ClNF-Ys in the regulation of watermelon immunity against diverse pathogens.


Introduction
Upon perception of internal and external cues, plants often initiate a complicated and fine-tuned transcriptional reprogramming network to modify the expression of specific sets of genes that are involved in growth, development, and stress response [1,2]. This transcriptional reprogramming of gene expression in plants requires the concerted action of epigenetic mechanisms (e.g., DNA methylation and histone modifications) and cooperative Watermelon (Citrullus lanatus L.), providing popular fresh fruit, is an important horticultural crop worldwide. Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the most devasting diseases that leads to great annual yield losses [59]; however, little is known about the genetic and molecular mechanisms governing the resistance against Fusarium wilt in watermelon. Previously, 19 ClNF-Y genes were identified and the expression of 13 ClNF-Y genes was found to be affected by drought and salt stress [60].
In the present study, we aimed to re-characterize the watermelon ClNF-Y family, analyze subcellular localization, assembly of ClNF-Y complexes, and expression changes in response to hormones and Fon, and explore the possible involvement of the ClNF-Y family in disease resistance. A total of 37 putative ClNF-Y complexes were identified. The expression levels of the ClNF-Y genes were changed after treatment with salicylic acid (SA), methyl jasmonate (MeJA), ABA, and ethylene precursor 1-aminocyclopropane-1carboxylate (ACC), or infection by Fon. Functional analyses through ectopic overexpression in Arabidopsis revealed that ClNF-YA2, -YA3, -YB1, -YB8, -YC2, and -YC4 play roles in disease resistance. The re-characterization of the ClNF-Y family, the definition of putative ClNF-Y complexes, and the identification of the functions of some ClNF-Y genes in disease resistance provide a basis from which to further investigate the biological functions and molecular mechanisms of the ClNF-Y genes in growth, development, and disease resistance against diverse pathogens in watermelon.

Evolution of the ClNF-Y Family
To gain insights into the expansion of the ClNF-Y family, the syntenic relationships between the ClNF-Y genes in the watermelon genome were examined. The results show that no tandem duplication event was detected but segmental duplication events for three gene pairs, ClNF-YA2/-YA3, ClNF-YA2/-YA5, and ClNF-YB2/-YB10, were identified in the ClNF-Y family ( Figure 3A), implying that the segmental duplication was the major force that drove the expansion of the CLNF-Y family. The nonsynonymous (Ka)/synonymous (Ks) ratios (Ka/Ks) of gene pairs ClNF-YA2/-YA3, ClNF-YA2/-YA5, and ClNF-YB2/-YB10 were estimated to be 0.2315, 0.3257, and 0.1122 (Table S1), respectively, indicating that these gene pairs evolved through purifying selection in watermelon. ClNF-YBs and ClNF-YC are formed by a minimum of three α-helices (α1, α2, and ( Figure S3B,C). Other conserved motifs were also identified in ClNF-YAs and ClNF ( Figure 2C), implying the diversity and complexity of the biochemical mechanisms o ClNF-Ys in watermelon.

Evolution of the ClNF-Y Family
To gain insights into the expansion of the ClNF-Y family, the syntenic relation between the ClNF-Y genes in the watermelon genome were examined. The results that no tandem duplication event was detected but segmental duplication events for gene pairs, ClNF-YA2/-YA3, ClNF-YA2/-YA5, and ClNF-YB2/-YB10, were identified i ClNF-Y family ( Figure 3A), implying that the segmental duplication was the major that drove the expansion of the CLNF-Y family. The nonsynonymous (Ka)/synonym (Ks) ratios (Ka/Ks) of gene pairs ClNF-YA2/-YA3, ClNF-YA2/-YA5, and ClNF-YB2/were estimated to be 0.2315, 0.3257, and 0.1122 (Table S1), respectively, indicating these gene pairs evolved through purifying selection in watermelon.  Interspecific comparative syntenic maps between the watermelon ClNF-Y genes and the NF-Y genes from Arabidopsis, rice, and cucumber were constructed to further elucidate the expansion mechanism of the ClNF-Y family. Interspecific collinearity analyses revealed that there were strong orthologs in the NF-Y families among watermelon, Arabidopsis, cucumber, and rice, and identified 19, 27, and 7 collinear gene pairs between watermelon and Arabidopsis, cucumber, and rice ( Figure 3B; Table S2). There are 23, 13, and 6 ClNF-Y genes with collinear orthologous gene pairs in cucumber, Arabidopsis, and rice genomes, respectively ( Figure 3B; Table S2). Notably, five ClNF-Y genes, ClNF-YA1, -YA6, -YB2, -YB7, and -YB10, have the same collinear orthologous gene pairs in cucumber, Arabidopsis, and rice genomes ( Figure 3B; Table S2), indicating that these five ClNF-Y genes may originate from common ancestors and were preserved during the evolution of plant species. With the exception of ClNF-YA7 and -YC6, the majority of the ClNF-Y genes have collinear orthologous gene pairs in the cucumber genome ( Figure 3B; Table S2), indicating that the NF-Y family is highly homologous among the cucurbit plants. The Ka/Ks ratios of the ClNF-Y collinear gene pairs identified in watermelon with those in rice, Arabidopsis, and cucumber are less than one (Table S2), suggesting that the watermelon ClNF-Y genes may have suffered strong purifying selective pressure during evolution.

Cis-Elements in Promoters of the ClNF-Y Genes
To gain insights into the responsiveness of the ClNF-Y genes, putative cis-elements in 1.5 Kb promoter regions of each ClNF-Y gene were analyzed using PlantCARE [63]. More than 2600 cis-elements belonging to 89 types were identified in promoters of the ClNF-Y genes, and apart from the common CAAT-box and TATA-box cis-elements, each of the ClNF-Y gene promoters contain an average of~45 cis-elements (Table S3). A large number of light-responsive cis-elements such as Box 4, G-box, GATA-motif, and GT1motif are present in almost all promoters of the ClNF-Y genes ( Figure S4). Importantly, different types of cis-elements responsible for plant growth and development and phytohormones, as well as abiotic and biotic stresses, were identified in the promoters of all of the ClNF-Y genes ( Figure 4). The cis-elements involved in plant growth and development included the development-related motif AAGAA, the senescence-related A-box, the vascular-specific element AC-II, the meristem expression motifs CAT-box and CCGTCC, the endosperm expression motif GCN4, and the zein metabolism regulation motif O2site ( Figure 4). The cis-elements involved in plant hormone response include the ABAresponsive element ABRE; the auxin-responsive elements AuxRR-core and TGA-element; gibberellin-responsive motifs CARE, GARE, P-box, and TATC; the ethylene-responsive element ERE; the SA-responsive elements TCA, SARE, and W box; and the MeJA-responsive motifs CGTCA and TGACG ( Figure 4). The cis-elements involved in abiotic and biotic stress response included ABRE4, ARE (anaerobic induction), AT-rich (defense activation), DRE core (dehydration-responsive), LTR (low-temperature responsive), MBS, MYC, MYB, MYB recognition, MYB-like, TC-rich (defense and stress responsive), WUN-motif (woundresponsive), box S (pathogen-inducible), STRE, and WRE3 ( Figure 4). In particular, 13 promoters of the ClNF-Y genes contain the SA-responsive TCA elements, 12 promoters harbor the MeJA-responsive element CGTCA-motif, 14 promoters carry the ethyleneresponsive element ERE, and 11 promoters possess the ABA-responsive element ABRE ( Figure 4). These data indicate the involvement of the ClNF-Y genes in plant growth, development, and stress response.   Table S3 for detail.

Subcellular Localization of the ClNF-Y Proteins
To explore the subcellular localization of the ClNF-Y proteins, agrobacteria carrying pCAMBIA1300-ClNF-YAs-GFP, pCAMBIA1300-ClNF-YBs-GFP, pCAMBIA1300ClNF-YCs-GFP, or pCAMBIA1300-GFP were infiltrated into the leaves of Nicotiana benthamiana plants expressing a red nuclear marker protein RFP-H2B [64]. The GFP signal from pCAMBIA1300-GFP-infiltrated leaves was distributed ubiquitously throughout the cells without specific compartmental localization ( Figure 5A). The GFP signal from pCAMBIA1300-ClNF-YAs-GFP-infiltrated leaves was mainly observed in the nucleus, which was co-localized with the known nucleus marker RFP-H2B ( Figure 5A). Similarly, a GFP signal from pCAMBIA1300-ClNF-YCs-GFP-infiltrated leaves was predominately seen in the nucleus, co-localized with RFP-H2B, except for pCAMBIA1300-ClNF-YC1-GFP, pCAMBIA1300-ClNF-YC4-GFP, pCAMBIA1300-ClNF-YC7-GFP, and pCAMBIA1300-ClNF-YC8-GFP, which were localized in the cells without specific compartmental localization ( Figure 5C). By contrast, the GFP signal from pCAMBIA1300-ClNF-YBs-GFP-infiltrated leaves was detected throughout the cellular compartments including the nucleus, except for ClNF-YB3, which was mainly localized in the nucleus ( Figure 5B). These data indicate that ClNF-YAs and ClNF-YCs are mainly targeted to the nucleus while ClNF-YBs are mostly localized in both nucleus and cytoplasm of the cells. Agrobacteria carrying pCAMBIA1300-ClNF-YAs-GFP, pCAM-BIA1300-ClNF-YBs-GFP, pCAMBIA1300-ClNF-YCs-GFP, or pCAMBIA1300-GFP were infiltrated into leaves of N. benthamiana plants expressing a known nucleus-localized marker protein RFP-H2B. At 48 h after agroinfiltration, the GFP signal was visualized under a confocal laser scanning microscope in a dark field for green fluorescence (left) and red fluorescence (middle left), a white field for cell morphology (middle right), and in combination (right). Scale bars, 20 μm. Experiments were performed three times with similar results.

Interactions between the ClNF-Y Subunits and Assembly of the ClNF-Y Complexes
It has been reported that Arabidopsis AtNF-YBs and AtNF-YCs interact with each other to form heterodimers [23,25]. To explore the assembly of the ClNF-Y complexes, the interactions between ClNF-YBs and ClNF-YCs were first examined through a yeast twohybrid (Y2H) system. However, some of the ClNF-YCs, such as ClNF-YC1, -YC2, and -YC4, showed autoactivation activity in Y2H assays even when a high concentration of AbA (500 ng/mL) was added to SD/-Trp plates ( Figure S5). Therefore, bimolecular fluorescent complimentary (BiFC) assays were performed to examine the interactions between ClNF-YBs and ClNF-YCs. The results show that a YFP signal was observed in leaves co- ClNF-YB subunits; (C) ClNF-YC subunits. Agrobacteria carrying pCAMBIA1300-ClNF-YAs-GFP, pCAMBIA1300-ClNF-YBs-GFP, pCAMBIA1300-ClNF-YCs-GFP, or pCAMBIA1300-GFP were infiltrated into leaves of N. benthamiana plants expressing a known nucleus-localized marker protein RFP-H2B. At 48 h after agroinfiltration, the GFP signal was visualized under a confocal laser scanning microscope in a dark field for green fluorescence (left) and red fluorescence (middle left), a white field for cell morphology (middle right), and in combination (right). Scale bars, 20 µm. Experiments were performed three times with similar results.

Expression Changes of ClNF-Y Genes in Response to Defense Hormones and a Fungal Pathogen
To explore the involvement of ClNF-Y genes in disease resistance, expression patterns were analyzed via reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) in watermelon plants after treatment with different stress hormones or infection by Fon, the causal agent of Fusarium wilt [59]. In plants treated with 1 mM SA, the expression of ClNF-YA4, -YB1, -YB4, -YB9, and -YC6 was upregulated, while the expression of ClNF-YA5, -YB6, -YB7, and -YC5 was downregulated compared to those in the untreated control plants ( Figure 7A). The expression of ClNF-YB7, -YB9, and -YC6 was upregulated, while the expression of ClNF-YA2, -YA3, -YA4, -YA5, -YB3, -YB5, -YC4, and -YC7 was downregulated in plants after treatment with 100 μM MeJA, compared to control plants ( Figure 7A). In plants treated with 100 μM ABA, the expression of ClNF-YA4, -YB6, -YB7, -YB9, and -YC7 was upregulated, while the expression of ClNF-YA1 and ClNF-YC5 was downregulated, compared to control plants ( Figure 7A). The expression of ClNF-YB3, -YB6, -YB7, and -YC7 was upregulated, while the expression of ClNF-YA4, and -YC1 was downregulated in plants after treatment with 100 μM ACC, compared to control plants ( Figure 7A). Overall, most of the ClNF-Y genes were upregulated by SA, ABA, and ACC, and downregulated by MeJA ( Figure 7A). Specifically, the expression of ClNF-YB9 was upregulated by SA, MeJA and ABA, while the expression of ClNF-YB7 was upregulated by MeJA, ABA, and ACC ( Figure 7A). These data indicate that the ClNF-Y genes are responsive to different stress hormones and thus may be involved in distinct hormone-mediated signaling pathways in respect of stress response.

Expression Changes of ClNF-Y Genes in Response to Defense Hormones and a Fungal Pathogen
To explore the involvement of ClNF-Y genes in disease resistance, expression patterns were analyzed via reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) in watermelon plants after treatment with different stress hormones or infection by Fon, the causal agent of Fusarium wilt [59]. In plants treated with 1 mM SA, the expression of ClNF-YA4, -YB1, -YB4, -YB9, and -YC6 was upregulated, while the expression of ClNF-YA5, -YB6, -YB7, and -YC5 was downregulated compared to those in the untreated control plants ( Figure 7A). The expression of ClNF-YB7, -YB9, and -YC6 was upregulated, while the expression of ClNF-YA2, -YA3, -YA4, -YA5, -YB3, -YB5, -YC4, and -YC7 was downregulated in plants after treatment with 100 µM MeJA, compared to control plants ( Figure 7A). In plants treated with 100 µM ABA, the expression of ClNF-YA4, -YB6, -YB7, -YB9, and -YC7 was upregulated, while the expression of ClNF-YA1 and ClNF-YC5 was downregulated, compared to control plants ( Figure 7A). The expression of ClNF-YB3, -YB6, -YB7, and -YC7 was upregulated, while the expression of ClNF-YA4, and -YC1 was downregulated in plants after treatment with 100 µM ACC, compared to control plants ( Figure 7A). Overall, most of the ClNF-Y genes were upregulated by SA, ABA, and ACC, and downregulated by MeJA ( Figure 7A). Specifically, the expression of ClNF-YB9 was upregulated by SA, MeJA and ABA, while the expression of ClNF-YB7 was upregulated by MeJA, ABA, and ACC ( Figure 7A). These data indicate that the ClNF-Y genes are responsive to different stress hormones and thus may be involved in distinct hormone-mediated signaling pathways in respect of stress response. YA1, -YA3, -YA6, -YB9, -YC1, -YC2, -YC3, -YC5, -YC6, -YC7, and -YC8 was remarkably upregulated by Fon, compared to mock-inoculated plants ( Figure 7C). Overall, most of the ClNF-Y genes were upregulated in the roots of the watermelon plants after Fon infection ( Figure 7C). These data indicate that most of the ClNF-Y genes respond to pathogen infection and thus may have functions in disease resistance against fusarium wilt.

Generation of ClNF-Y-Overexpressing Arabidopsis Lines and the Involvement of ClNF-Y in Growth and Development
To explore the biological functions of the ClNF-Y genes, 10 genes (ClNF-YA2, -YA3, -YB1, -YB7, -YB8, -YB9, -YC1, -YC2, -YC4, and -YC7), based on the assembly of ClNF-Y complexes ( Figure 6) and the expression patterns (Figure 7), were selected to generate overexpression transgenic Arabidopsis lines through the floral dip method [66]. After hygromycin-resistance screening and genetic analyses, two homozygous transgenic lines with single-copy for each of the ClNF-Y genes (T3 generation) and similar expression levels of the transgenes were chosen for further studies. RT-qPCR analyses indicated that the ClNF-Y genes were transcribed normally in the transgenic Arabidopsis lines ( Figure S8). Before bolting, the ClNF-Y-overexpressing Arabidopsis plants grew normally and were indistinguishable from the wild-type (WT) plants in terms of growth and morphological phenotype ( Figure 8A). The ClNF-YB1-OE, ClNF-YB8-OE, ClNF-YB9-OE, ClNF-YC2-OE, and ClNF-YC7-OE plants flowered earlier by 2-4 d compared with the WT plants; the ClNF-YA2-OE, ClNF-YA3-OE, ClNF-YB7-OE, ClNF-YC1-OE, and ClNF-YC4-OE plants showed similar flowering to the WT plants ( Figure 8B). After bolting, six-week-old ClNF-YB1-OE, ClNF-YB8-OE, and ClNF-YC7-OE plants were taller than the WT plants, while the plant heights of the other transgenic lines were comparable to the WT plants ( Figure 8C,D). These data indicate that ClNF-YB1, -YB8, -YB9, -YC2, and -YC7 play roles in flowering, and that ClNF-YB1, -YB8, and -YC7 also function in vegetative growth.  Figure 6) and the expression patterns (Figure 7), were selected to generate overexpression transgenic Arabidopsis lines through the floral dip method [66]. After hy gromycin-resistance screening and genetic analyses, two homozygous transgenic line with single-copy for each of the ClNF-Y genes (T3 generation) and similar expression lev els of the transgenes were chosen for further studies. RT-qPCR analyses indicated that the ClNF-Y genes were transcribed normally in the transgenic Arabidopsis lines ( Figure S8) Before bolting, the ClNF-Y-overexpressing Arabidopsis plants grew normally and were indistinguishable from the wild-type (WT) plants in terms of growth and morphologica phenotype ( Figure 8A)

Functions of the ClNF-Y Genes in Disease Resistance
To explore the possible functions of the ClNF-Y genes in disease resistance, the ClNF-Y-overexpressing Arabidopsis lines were assessed for their resistance phenotype against Botrytis cinerea, a necrotrophic fungus causing grey mold disease. When fully expanded leaves from four-week-old plants were inoculated with a drop of 3 µL spore suspension (2 × 10 5 spores/mL), typical B.-cinerea-caused water-soaked necrotic lesions appeared at 2 dpi ( Figure 9A). In repeated assays, the necrotic lesions on the detached leaves of the ClNF-YA2-OE and ClNF-YC2-OE plants were significantly larger, resulting in increases of approximately 12%, and 17%, respectively, while the necrotic lesions on the detached leaves of the ClNF-YB8-OE plants were remarkably smaller, leading to a reduction of 32% in comparison to those on the WT leaves at 3 dpi ( Figure 9A,B). Without infection of B. cinerea, the expression of AtPR5 in ClNF-YC2-OE plants were markedly downregulated, while no significant change in the expression of AtPR1 in ClNF-YA2-OE, ClNF-YB8-OE, and ClNF-YC2-OE plants and AtPR5 in ClNF-YA2-OE and ClNF-YB8-OE plants was observed ( Figure 9C). However, the expression levels of AtPR1 and AtPR5 were significantly downregulated in ClNF-YA2-OE and ClNF-YC2-OE plants but upregulated in ClNF-YB8-OE plants after infection of B. cinerea ( Figure 9C). B. cinerea-caused necrotic lesions on the detached leaves of the ClNF-YA3-OE, ClNF-YB1-OE, ClNF-YB7-OE, ClNF-YB9-OE, ClNF-YC1-OE, ClNF-YC4-OE, and ClNF-YC7-OE plants were comparable to those on the WT leaves ( Figure 9A,B). These data suggest that ClNF-YB8 plays a positive role while ClNF-YA2 and -YC2 function negatively in terms of disease resistance against B. cinerea in transgenic Arabidopsis plants.  The function of ClNF-Y genes in disease resistance was further investigated through assessing the resistance phenotype of the ClNF-Y-overexpressing transgenic Arabidopsis plants against Pseudomonas syringae pv. tomato (Pst) DC3000, a hemibiotrophic bacterial pathogen causing leaf spot disease. When the Arabidopsis leaves were inoculated by injecting a bacterial inoculum of Pst DC3000, typical yellowing symptoms were observed on the inoculated leaves at 4 dpi ( Figure 10A). Compared with those in the inoculated WT leaves, diseases on the inoculated leaves of the ClNF-YA3-OE, ClNF-YB1-OE, and ClNF-YC4-OE plants were reduced and these leaves supported less bacterial growth, resulting in decreases of 1.37, 1.26, and 1.22 orders of magnitude at 2 dpi ( Figure 10A,B). By contrast, diseases on the inoculated leaves of ClNF-YA2-OE and ClNF-YB8-OE plants were much more severe, showing larger yellowing and necrotic areas, and these leaves supported more bacterial growth, leading to increases of 1.15 and 1.29 orders of magnitude at 2 dpi, as compared with those in the inoculated WT leaves ( Figure 10A,B). Without infection of Pst DC3000, the expression of AtPR1 and AtPR5 in ClNF-YA2-OE, ClNF-YA3-OE, ClNF-YB1-OE, ClNF-YB8-OE, and ClNF-YC4-OE plants was not affected, except that the expression of AtPR5 in ClNF-YA3-OE plants were markedly downregulated ( Figure 10C). However, the expression levels of AtPR1 and AtPR5 were significantly upregulated in ClNF-YA3-OE, ClNF-YB1-OE, and ClNF-YC4-OE plants but downregulated in ClNF-YA2-OE and ClNF-YB8-OE plants after infection of Pst DC3000 ( Figure 10C). In addition, disease symptoms on the inoculated leaves of ClNF-YB7-OE, ClNF-YB9-OE, ClNF-YC1-OE, ClNF-YC2-OE, and ClNF-YC7-OE plants were indistinguishable from those on the inoculated WT leaves ( Figure 10A,B). These data indicate that ClNF-YA3, -YB1, and -YC4 positively regulate while ClNF-YA2 and -YB8 negatively modulate the resistance of transgenic Arabidopsis plants against Pst DC3000.

Discussion
Unlike those in mammals and yeasts, the subunits of plant NF-Y complexes are encoded by relatively small gene families [20]. A Previous study has identified 19 ClNF-Y genes in the watermelon genome [60]. In the present study, further bioinformatics analyses identified a total 25 ClNF-Y genes in watermelon, among which 7 encode for ClNF-YAs, 10 for ClNF-YBs, and 8 for ClNF-YCs (Table 1), similar to those in cucumber [12], but fewer than those in Arabidopsis (36 AtNF-Ys), rice (34 OsNF-Ys), and tomato (59 SlNF-Ys) [10,11,16]. The ClNF-YA genes showed a highly complicated intron-exon organization with 4-7 introns, while the ClNF-YB and ClNF-YC genes exhibited variable intron-exon organizations ( Figure 2B). Specifically, more than half of the ClNF-YB (5/10) and ClNF-YC (5/8) genes were intronless ( Figure 2B), which is consistent with a universal feature of NF-YB and NF-YC genes in other plant species including cucumber [12]. Phylogenetic tree analysis revealed that the watermelon ClNF-Y proteins were closely related to those in Arabidopsis (Figure 1). These characteristics in phylogenetic relationships and gene structure imply the conserved feature of the evolution of the NF-Y families in plants. In the ClNF-Y family, only three segmentally duplicated genes were identified, and no tandemly duplicated gene was detected ( Figure 3A), suggesting that segmental duplication is the major force driving the expansion of the ClNF-Y family in watermelon. This differs from that of the rice OsNF-Y and cucumber CsNF-Y families, whose expansions were driven by both segmental and tandem duplication events [11,12]. Furthermore, analyses of the interspecific syntenic relationship and the Ka/Ks ratios of the collinear ClNF-Y gene pairs with other plant species ( Figure 3B; Tables S1 and S2) revealed that purifying selective pressure may have been a strong driving force in the evolution of the ClNF-Y family in watermelon.
The three subunits (NF-YA, NF-YB, and NF-YC) of the NF-Y complexes are generally recognized by the presence of conserved domains responsible for the interaction between the subunits and DNA binding to the CCAAT cis-element in the promoters of the target genes [6]. The ClNF-YAs contain NF-YB/YC interaction and DNA-binding domains, while the ClNF-YBs and ClNF-YCs harbor the HFM domains ( Figure 2C and S3). In addition, other conserved motifs were identified in ClNF-YAs and ClNF-YCs ( Figure 2C). The structural features confer the basis for the subcellular localization, interaction, and biochemical activities of the ClNF-YAs, ClNF-YBs, and ClNF-YCs. For example, the ClNF-YAs and ClNF-YCs were predominately localized in nucleus ( Figure 5A,C), which is consistent with the common knowledge that NF-YAs and NF-YCs contain NLSs and thus are generally localized to the nucleus [7,8,20,23]. Notably, ClNF-YC1, -YC4, -YC7, and -YC8 were found to localize in both the nucleus and cytoplasm of the cells, although they harbor NLSs similar to ClNF-YC2, -YC3, -YC5, and -YC6, which were predominately localized in nucleus, in the BiFC assays ( Figure 5C). Because the NF-YBs and NF-YCs generally heterodimerize in the cytoplasm and then translocate into the nucleus [22,25,26], it is speculated that the difference in subcellular localization of ClNF-YCs may be due to their interactions with N. benthamiana NF-YBs. Unlike NF-YAs and NF-YCs, NF-YBs are not localized in the nucleus due to the lack of NLSs [19]. The majority of the ClNF-YBs were found to be localized in both the nucleus and cytoplasm of the cells without specific compartments ( Figure 5B); however, ClNF-YB3 seemed to be localized in the nucleus ( Figure 6B). Similar phenomena were also observed for cassava MeNF-YB11 and MeNF-YB16, and Picea wilsonii PwNF-YB3, which were mainly localized in the nucleus [58,67]. It is known that interactions and the formation of heterodimers with NF-YCs are essential for the translocation of NF-YBs from the cytoplasm into the nucleus [25,65]. In this regard, the nuclear localization of ClNF-YB3 ( Figure 5B) may be due to the formation of heterodimers through interacting with unknown N. benthamiana NF-YCs in planta.
The functions of NF-Ys in plant growth and development have been well documented [6,28]. Bioinformatics analysis identified plenty light-responsive, growth, and development-associated cis-elements in promoters of the ClNF-Y genes (Figures 4 and S4), implying their involvement in the regulation of growth and development. This is directly supported by the observations that the ClNF-YB1-OE, ClNF-YB8-OE, ClNF-YB9-OE, ClNF-YC2-OE, and ClNF-YC7-OE plants showed an earlier flowering phenotype and that the ClNF-YB1-OE, ClNF-YB8-OE, and ClNF-YC7-OE plants grew taller after bolting (Figure 8). ClNF-YB1, -YB8, and -YC2 are phylogenetically related to AtNF-YB3, -YB9, and -YC2, respectively (Figure 1), which play positive roles in the promotion of flowering in Arabidopsis [68,69]. Notably, ClNF-YB1, -YB8, and -YC7 exhibited a pleiotropic effect on flowering and growth in transgenic Arabidopsis. Moreover, 5 of the 10 ClNF-Y genes selected for functional studies in transgenic Arabidopsis conferred an earlier flowering phenotype, indicating the wide involvement of the ClNF-Y family in the regulation of flowering time. Detailed examination of the phenotypes (e.g., seed setting, size, and weight) of the ClNF-Y-overexpressing Arabidopsis lines, especially ClNF-YB1-OE, ClNF-YB8-OE, and ClNF-YC7-OE lines, will provide further understanding on the functions and mechanism of the ClNF-Y genes in growth and development.
The plant NF-Y family has also been implicated in abiotic and biotic stress responses [6,28]. The fact that a large number of hormone-and stress-responsive ciselements are present in the promoter regions ( Figure 4) suggests the involvement of the ClNF-Y family in stress response. In particular, the ClNF-YB7 and -YC7 promoters harbor ABA-responsive element ABRE (Figure 4), and accordingly, the expression of ClNF-YB7 and -YC7 was upregulated by ABA ( Figure 7A), implying their involvement in abiotic stress response. This is partially supported by the observation that Arabidopsis AtNF-YC2, closely related to ClNF-YC7 (Figure 1), has been implicated in the regulation of stress genes in Arabidopsis [65]. However, the presence of the SA-responsive TCA element in 13 promoters of the ClNF-Y genes, the MeJA-responsive element CGTCA-motif in 12 promoters, and the ethylene-responsive element ERE in 14 promoters (Figure 4), implies that these ClNF-Y genes may participate in the SA-, JA-, and ethylene-mediated signaling pathways and thus play roles in disease resistance in watermelon. The presence of defense-hormone-responsive cis-elements in the promoters seems to be consistent with the expression changes in the ClNF-Y genes in watermelon plants after treatment with SA, MeJA, or ACC. For instance, the ClNF-YB9 promoter contains three MeJA-responsive CGTCA-motifs ( Figure 4); accordingly, the expression of ClNF-YB9 was induced by MeJA ( Figure 7A). Similarly, the ClNF-YB6 and -YB7 promoters harbored three and two ethyleneresponsive ERE elements, respectively (Figure 4), and their expression was strongly upregulated by ACC ( Figure 7A). Furthermore, the majority of the ClNF-Y genes were induced by Fon, and the pathogen-induced expression was highly evident in roots ( Figure 7C). Among these pathogen-inducible ClNF-Y genes, the ClNF-YA1, -YB8, and -YB9 promoters harbor a pathogen-inducible Box S element (Figure 4), which is known to confer a high level of expression of the target genes in response to elicitors, oomycetes, and bacterial pathogens [70][71][72]. Functional studies in transgenic Arabidopsis revealed that 6 ClNF-Y genes play a role in disease resistance (Figures 9 and 10). Specifically, ClNF-YA2 and -YC2 negatively regulated while ClNF-YB8 positively regulated resistance against B. cinerea in transgenic Arabidopsis plants (Figure 9). ClNF-YA2 and -YB8 negatively regulated while ClNF-YA3, -YB1, and -YC4 positively modulated resistance against Pst DC3000 in transgenic Arabidopsis plants ( Figure 10). The alterations in resistance against B. cinerea and Pst DC3000 were accompanied with the changes in the pathogen-induced expression of defense genes in ClNF-YA2-OE, ClNF-YA3-OE, ClNF-YB1-OE, ClNF-YB8-OE, ClNF-YC2-OE, and ClNF-YC4-OE plants (Figures 9 and 10). Notably, overexpression of ClNF-YA2 in transgenic Arabidopsis resulted in attenuated resistance against both B. cinerea and Pst DC3000; however, overexpression of ClNF-YB8 led to opposite functions in resistance against these two pathogens, e.g., enhanced resistance against B. cinerea but attenuated resistance against Pst DC3000 (Figures 9 and 10). Generally, the defense response against (hemi)biotrophic pathogens such as Pst DC3000 is modulated through SA signaling, while resistance against necrotrophic pathogens like B. cinerea is regulated by JA/ET signaling [73,74]. There are both antagonistic and synergistic interactions between the SA and JA/ET signaling pathways to enable plants to activate appropriate defense responses against different invading pathogens [73,75,76]. In this regard, it is therefore likely that ClNF-YA2 and -YB8 function in disease resistance through different mechanisms. Further characterization of the target genes will provide insights into the molecular mechanisms by which key subunits or the ClNF-Y complexes regulate disease resistance in plants. Furthermore, the fact that 6 of the 10 functionally studied ClNF-Y genes played roles in disease resistance (Figures 9 and 10) indicates the importance of the ClNF-Y family in plant disease resistance. This is similar to the recent observation that overexpression of 3 out of 11 rice OsNF-YA genes significantly affected susceptibility to two viral pathogens [55]. Therefore, further functional studies on the remaining ClNF-Y genes will be helpful to provide comprehensive understanding of the involvement of the ClNF-Y family in disease resistance. , or an equal volume of solution containing only 0.1% ethanol and 0.02% Tween20 as controls. Pathogen inoculation was performed on three-week-old watermelon plants by the root-dipping inoculation method as previously described [77]. Spore inoculum of Fon race 1 strain ZJ1 (1 × 10 7 spores/mL) was prepared as previously described [78]. The main roots of the watermelon plants were cut up to one-third, and then dipped for 15 min in spore inoculum of Fon or in medium broth as mock-inoculated controls. The inoculated plants were replanted in soil and allowed to grow in the same growth room as described above. Root and leaf samples were collected at indicated time points after treatment/inoculation, frozen in liquid nitrogen, and stored at −80 • C until use.

Identification and Bioinformatics Analysis of the Watermelon ClNF-Y Family
Arabidopsis AtNF-Ys protein sequences were downloaded from TAIR (https://www. arabidopsis.org, accessed on 11 May 2022) based on a previous report [10] and were used as queries to search via the BLASTp program for putative ClNF-Y genes and proteins in the watermelon genome in the Cucurbit Genomics Databases (http://cucurbitgenomics.org/ organism/21 for 97,103 v2 and http://cucurbitgenomics.org/organism/4 for cv. Charleston Gray, accessed on 11 May 2022) [61,62]. The putative ClNF-Ys protein sequences were examined by domain analysis programs PFAM (http://pfam.sanger.ac.uk/, accessed on 13 May 2022) (PF02045 and PF00808) and SMART (http://smart.emblheidelberg.de/, accessed on 13 May 2022) with default cutoff parameters. The protein properties of ClNF-Ys, such as the number of amino acids, molecular weights, and isoelectric points (pI) were predicted on the ExPASy Proteomics Server (http://expasy.org/, accessed on 15 May 2022). Sequence alignment was carried out using the Clustal X1.8 program, and a phylogenetic tree was constructed using the pairwise gap deletion option, Poisson model, and 1000 bootstrap replicates in MEGA version 7.0 (https://www.megasoftware.net, accessed on 20 May 2022). Putative conserved motifs in the ClNF-Y proteins were characterized using the Multiple Em for Motif Elicitation program (MEME, http://www.meme.sdsc.edu/meme/meme.html, accessed on 20 May 2022) with optimized parameter settings: repetition number, any; minimum motif width, 6; maximum motif width, 50; maximum number of motifs, 20 [79].
A gene structure featuring introns and exons in the predicted ClNF-Y genes was constructed using Gene Structure Display Server 2.0 (GSDS) (http://gsds.cbi.pku.edu.cn/, accessed on 21 May 2022) [80]. The MCScanX algorithm with default parameters [81] was used to scan orthologous regions containing the watermelon ClNF-Y genes, and the corresponding plot was created using Dual Synteny Plot for MCscanX in TBtools software version 1.1044 [82]. The chromosomal localization of the ClNF-Y genes was obtained in the watermelon genome database (http://cucurbitgenomics.org/organism/21, accessed on 23 May 2022) and visualized using MapChart software (https://www.wur.nl/en/ show/Mapchart.htm, accessed on 23 May 2022) [83]. The synteny relationships of the orthologous NF-Y genes between watermelon and other selected species (Arabidopsis, rice, and cucumber) were visualized using the Advance Circos package of TBtools [83]. DnaSP software version 6 was used to calculate the nonsynonymous (Ka)/synonymous (Ks) values of the duplicated ClNF-Y gene pairs [84]. The Plant CARE database (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 25 May 2022) was employed to predict the putative cis-elements in the 1500 bp promoter regions of the ClNF-Y genes [63].

Cloning of ClNF-Y Genes
Total RNA was extracted using Trizol reagent and treated with RNase-free DNase (Takara, Tokyo, Japan) according to the manufacturer's instructions. First-strand cDNA was synthesized using AMV reverse transcriptase (Takara, Tokyo, Japan) with oligo d(T) primer according to the manufacturer's instructions. The coding sequences for ClNF-Y genes were amplified using gene-specific primers (Table S4) and cloned into pMD19-T vector (Takara, Tokyo, Japan) via T/A cloning, yielding pMD19-ClNF-YAs, pMD19-ClNF-YBs, and pMD19-ClNF-YCs, which were confirmed by sequencing.

Subcellular Localization Assays
The coding sequences of the ClNF-Y genes were amplified using gene-specific primers (Table S4) and inserted into pCAMBIA1300s, generating pCAMBIA1300s-ClNF-Ys-GFPs, which were then transformed into Agrobacterium tumefaciens strain GV3101. Agrobacteria carrying pCAMBIA1300s-ClNF-Ys-GFP or pCAMBIA1300s-GFP were infiltrated into leaves of N. benthamiana plants expressing the RFP-H2B marker [64]. The GFP signal was excited at 488 nm and detected under a Zeiss LSM780 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) using a 500-530 nm emission filter, at 48 h after agroinfiltration.

RT-qPCR Assays
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and then reverse-transcribed into cDNA using the HiScript QRT SuperMix kit (Vazyme, Nanjing, China). Each qPCR contained 12.5 µL of AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China), 0.1 µg of cDNA, and 7.5 pmol of each gene-specific primer (Table S4) in a final volume of 25 µL. The qPCR was performed in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Watermelon ClGAPDH was used as an internal control and relative gene expression levels were calculated using the 2 − CT method. Data were normalized with those in mock-treated or mock-inoculated plants at each time point. Primers used are listed in Table S4.

Generation and Characterization of ClNF-Y-Overexpressing Transgenic Lines
Arabidopsis transformation with agrobacteria carrying pCAMBIA1300s-ClNF-Ys-GFP was performed using the floral dip method [66]. Putative positive transgenic plants from T1 seeds were selected on 1/2 MS medium containing 50 µg/mL hygromycin. Single-copy transgenic lines and homozygous lines were obtained by screening for a 3:1 segregation ratio of hygromycin-resistant character and 100% hygromycin-resistant phenotype in T2 and T3 generations on 1/2 MS medium supplemented with 50 µg/mL hygromycin, respectively.

Disease Assays
Disease assays with B. cinerea were performed as previously described [85]. B. cinerea was grown on 2 × V 8 (36% V 8 juice, 0.2% CaCO 3 , 2% agar) medium at 25 • C for 8~10 d, and spores were collected, and then resuspended in a 4% maltose and 1% peptone buffer to a final concentration of 2 × 10 5 spores/mL. Fully expanded leaves were detached from four-week-old Arabidopsis plants and inoculated by dropping 3 µL spore suspension. Disease was estimated by measuring the lesion sizes.
P. syringae pv. tomato DC3000 was grown in King's B broth and collected by centrifugation, followed by re-suspending in 10 mM MgCl 2 solution to OD 600 = 0.002. Inoculation was performed by hand infiltration using 1 mL syringes without needles into rosette leaves of four-week-old Arabidopsis plants, as described previously [86]. Leaf discs from inoculated leaves were collected and homogenized in 10 mM MgCl 2 to quantify in planta bacterial growth.

Statistical Analysis
All the experiments were performed independently at least three times. The data obtained were subjected to statistical analysis according to Student's t-test, and the probability values of p < 0.05 or p < 0.01 were considered as significant difference between different treatments.

Conclusions
In the present study, the watermelon ClNF-Y family was re-characterized and a total of 25 family members (7 ClNF-YAs, 10 CLNF-YBs, and 8 ClNF-YCs) were identified, further enlarging the family by adding 6 new members [60]. Structural features of genes and proteins, phylogenetic and syntenic relationships, cis-elements in promoters, subcellular localization, assembly of the ClNF-Y complexes, expression changes in response to defense hormones and pathogen infection, and putative functions in disease resistance were comprehensively investigated. A total of 37 putative ClNF-Y complexes that were assembled by ClNF-YA1, -YA2, -YA3, and -YA7 with diverse ClNF-YB/-YC heterodimers were identified. Expression analysis revealed that most of the ClNF-Y genes responded with distinct patterns to defense hormones and infection of a vascular-infecting pathogen, F. oxysporum f. sp. niveum. Functional studies in transgenic Arabidopsis revealed that 6 ClNF-Y genes (ClNF-YA2, -YA3, -YB1, -YB8, -YC2, and -YC4) played roles in disease resistance. It should be noted, however, that the functional analysis in the present study was performed via ectopic overexpression of the ClNF-Y genes in Arabidopsis, and the intrinsic functions of the ClNF-Y genes, especially those having a disease resistance function in transgenic Arabidopsis, need to be further investigated in watermelon disease resistance through overexpression and CRISPR/Cas9-based knockout approaches. The re-characterization of the ClNF-Y family provides a foundation from which to investigate the biological function of ClNF-Y genes in terms of growth, development, and stress response in watermelon, and the identification of the functions of some ClNF-Y genes in disease resistance enables further exploration of the molecular mechanism of ClNF-Ys in regulating watermelon disease resistance.