Chromosome-level pepino genome provides insights into genome evolution and anthocyanin biosynthesis in Solanaceae.

Pepino (Solanum muricatum, 2n = 2x = 24), a member of the Solanaceae family, is an important globally grown fruit. Herein, we report high-quality, chromosome-level pepino genomes. The 91.67% genome sequence is anchored to 12 chromosomes, with a total length of 1.20 Gb and scaffold N50 of 87.03 Mb. More than half the genome comprises repetitive sequences. In addition to the shared ancient whole-genome triplication (WGT) event in eudicots, an additional new WGT event was present in the pepino. Our findings suggest that pepinos experienced chromosome rearrangements, fusions, and gene loss after a WGT event. The large number of gene removals indicated the instability of Solanaceae genomes, providing opportunities for species divergence and natural selection. The paucity of disease-resistance genes (NBS) in pepino and eggplant has been explained by extensive loss and limited generation of genes after WGT events in Solanaceae. The outbreak of NBS genes was not synchronized in Solanaceae species, which occurred before the Solanaceae WGT event in pepino, tomato, and tobacco, whereas it was almost synchronized with WGT events in the other four Solanaceae species. Transcriptome and comparative genomic analyses revealed several key genes involved in anthocyanin biosynthesis. Although an extra WGT event occurred in Solanaceae, CHS genes related to anthocyanin biosynthesis in grapes were still significantly expanded compared with those in Solanaceae species. Proximal and tandem duplications contributed to the expansion of CHS genes. In conclusion, the pepino genome and annotation facilitate further research into important gene functions and comparative genomic analysis in Solanaceae.

Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis.

Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility.

Whole-Genome Sequence of Synthesized Allopolyploids in Cucumis Reveals Insights into the Genome Evolution of Allopolyploidization.

The importance of allopolyploidy in plant evolution has been widely recognized. The genetic changes triggered by allopolyploidy, however, are not yet fully understood due to inconsistent phenomena reported across diverse species. The construction of synthetic polyploids offers a controlled approach to systematically reveal genomic changes that occur during the process of polyploidy. This study reports the first fully sequenced synthetic allopolyploid constructed from a cross between Cucumis sativus and C. hystrix, with high-quality assembly. The two subgenomes are confidently partitioned and the C. sativus-originated subgenome predominates over the C. hystrix-originated subgenome, retaining more sequences and showing higher homeologous gene expression. Most of the genomic changes emerge immediately after interspecific hybridization. Analysis of a series of genome sequences from several generations (S0, S4-S13) of C. ×hytivus confirms that genomic changes occurred in the very first generations, subsequently slowing down as the process of diploidization is initiated. The duplicated genome of the allopolyploid with double genes from both parents broadens the genetic base of C. ×hytivus, resulting in enhanced phenotypic plasticity. This study provides novel insights into plant polyploid genome evolution and demonstrates a promising strategy for the development of a wide array of novel plant species and varieties through artificial polyploidization.

Complete chloroplast genome sequencing and comparative analysis reveals changes to the chloroplast genome after allopolyploidization in Cucumis.

Allopolyploids undergo "genomic shock" leading to significant genetic and epigenetic modifications. Previous studies have mainly focused on nuclear changes, while little is known about the inheritance and changes of organelle genome in allopolyploidization. The synthetic allotetraploid Cucumis ×hytivus, which is generated via hybridization between C. hystrix and C. sativus, is a useful model system for studying cytonuclear variation. Here, we report the chloroplast genome of allotetraploid C. ×hytivus and its diploid parents via sequencing and comparative analysis. The size of the obtained chloroplast genomes ranged from 154 673 to 155 760 bp, while their gene contents, gene orders, and GC contents were similar to each other. Comparative genome analysis supports chloroplast maternal inheritance. However, we identified 51 indels and 292 SNP genetic variants in the chloroplast genome of the allopolyploid C. ×hytivus relative to its female parent C. hystrix. Nine intergenic regions with rich variation were identified through comparative analysis of the chloroplast genomes within the subgenus Cucumis. The phylogenetic network based on the chloroplast genome sequences clarified the evolution and taxonomic position of the synthetic allotetraploid C. ×hytivus. The results of this study provide us with an insight into the changes of organelle genome after allopolyploidization, and a new understanding of the cytonuclear evolution.

Global Profiling of lncRNAs Expression Responsive to Allopolyploidization in Cucumis.

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various biological processes. However, the presence of lncRNAs and how they function in plant polyploidy are still largely unknown. Hence, we examined the profile of lncRNAs in a nascent allotetraploid Cucumis hytivus (S14), its diploid parents, and the F1 hybrid, to reveal the function of lncRNAs in plant-interspecific hybridization and whole genome duplication. Results showed that 2206 lncRNAs evenly transcribed from all 19 chromosomes were identified in C. hytivus, 44.6% of which were from intergenic regions. Based on the expression trend in allopolyploidization, we found that a high proportion of lncRNAs (94.6%) showed up-regulated expression to varying degrees following hybridization. However, few lncRNAs (33, 2.1%) were non-additively expressed after genome duplication, suggesting the significant effect of hybridization on lncRNAs, rather than genome duplication. Furthermore, 253 cis-regulated target genes were predicted for these differentially expressed lncRNAs in S14, which mainly participated in chloroplast biological regulation (e.g., chlorophyll synthesis and light harvesting system). Overall, this study provides new insight into the function of lncRNAs during the processes of hybridization and polyploidization in plant evolution.

Nuclear-Cytoplasmic Coevolution Analysis of RuBisCO in Synthesized Cucumis Allopolyploid.

Allopolyploids are often faced with the challenge of maintaining well-coordination between nuclear and cytoplasmic genes inherited from different species. The synthetic allotetraploid Cucumis × hytivus is a useful model to explore cytonuclear coevolution. In this study, the sequences and expression of cytonuclear enzyme complex RuBisCO as well as its content and activity in C. × hytivus were compared to its parents to explore plastid-nuclear coevolution. The plastome-coded rbcL gene sequence was confirmed to be stable maternal inheritance, and parental copy of nuclear rbcS genes were both preserved in C. × hytivus. Thus, the maternal plastid may interact with the biparentally inherited rbcS alleles. The expression of the rbcS gene of C-homoeologs (paternal) was significantly higher than that of H-homoeologs (maternal) in C. × hytivus (HHCC). Protein interaction prediction analysis showed that the rbcL protein has stronger binding affinity to the paternal copy of rbcS protein than that of maternal copy in C. × hytivus, which might explain the transcriptional bias of the rbcS homoeologs. Moreover, both the activity and content of RuBisCO in C. × hytivus showed mid-parent heterosis. In summary, our results indicate a paternal transcriptional bias of the rbcS genes in C. × hytivus, and we found new nuclear-cytoplasmic combination may be one of the reasons for allopolyploids heterosis.

Effect of silver nanoparticles on phenolic compounds production and biological activities in hairy root cultures of Cucumis anguria.

The present study describes the elicitor effect of silver ion (Ag+) and biologically synthesized silver nanoparticles (AgNPs) to enhance the biomass accumulation and phenolic compound production as well as biological activities (antioxidant, antimicrobial and anticancer) in genetically transformed root (hairy root) cultures of Cucumis anguria. The biomass of hairy root cultures was significantly increased by AgNPs whereas decreased in Ag+ elicitation at 1 and 2 mg/L. AgNPs-elicited hairy roots produced a significantly higher amount of individual phenolic compounds (flavonols, hydroxycinnamic and hydroxybenzoic acids), total phenolic and flavonoid contents than Ag+-elicited hairy roots. Moreover, antioxidant, antimicrobial and anticancer activities were significantly higher following AgNPs-elicitation compared with that in Ag+-elicited hairy roots. We suggest that AgNPs could be an efficient elicitor in hairy root cultures to increase the phytochemical production.

Identification of all homoeologous chromosomes of newly synthetic allotetraploid Cucumis × hytivus and its wild parent reveals stable subgenome structure.

Allopolyploidy and homoeologous recombination are two important processes in reshaping genomes and generating evolutionary novelties. Newly formed allopolyploids usually display chromosomal perturbations as a result of pairing errors at meiosis. To understand mechanisms of stabilization of allopolyploid species derived from distant chromosome bases, we investigated mitotic stability of a synthetic Cucumis allotetraploid species in relation to meiosis chromosome behavior. The Cucumis × hytivus is an allotetraploid synthesized from interspecific hybridization between cucumber (Cucumis sativus, 2n = 14) and its wild relative Cucumis hystrix (2n = 24) followed by spontaneous chromosome doubling. In the present study, we analyzed the wild parent C. hystrix and the latest generation of C. hytivus using GISH (genomic in situ hybridization) and cross-species FISH (fluorescence in situ hybridization). The karyotype of C. hystrix was constructed with two methods using cucumber fosmid clones and repetitive sequences. Using repeat-element probe mix in two successive hybridizations allowed for routine identification of all 19 homoeologous chromosomes of allotetraploid C. hytivus. No aneuploids were identified in any C. hytivus individuals that were characterized, and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid. Meiotic irregularities, such as homoeologous pairing, were frequently observed, resulting in univalent and intergenomic multivalent formation. The relatively stable chromosome structure of the synthetic Cucumis allotetraploid may be explained by more deleterious chromosomal viable gametes compared with other allopolyploids. The knowledge of genetic and genomic information of Cucumis allotetraploid species could provide novel insights into the establishment of allopolyploids with different chromosome bases.

Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis.

Ribosomal DNAs are useful cytogenetic markers for chromosome analysis. Studies investigating site numbers and distributions of rDNAs have provided important information for elucidating genome organization and chromosomal relationships of many species by fluorescence in situ hybridization. But relevant studies are scarce for species of the genus Cucumis, especially in wild species. In the present study, FISH was conducted to investigate the organization of 45S and 5S rDNA among 20 Cucumis accessions, including cultivars and wild accessions. Our results showed that the number of 45S rDNA sites varied from one to five pairs in different accessions, and most of these sites are located at the terminal regions of chromosomes. Interestingly, up to five pairs of 45S rDNA sites were observed in C. sativus var. sativus, the species which has the lowest chromosome number, i.e., 2n = 14. Only one pair of 5S rDNA sites was detected in all accessions, except for C. heptadactylus, C. sp, and C. spp that had two pairs of 5S rDNA sites. The distributions of 5S rDNA sites showed more variation than 45S rDNA sites. The phylogenetic analysis in this study showed that 45S and 5S rDNA have contrasting evolutionary patterns. We find that 5S rDNA has a polyploidization-related tendency towards the terminal location from an interstitial location but maintains a conserved site number, whereas the 45S rDNA showed a trend of increasing site number but a relatively conserved location.

Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

Molecular Cytogenetic Analysis of Cucumis Wild Species Distributed in Southern Africa: Physical Mapping of 5S and 45S rDNA with DAPI.

Wild Cucumis species have been divided into Australian/Asian and African groups using morphological and phylogenetic characteristics, and new species have been described recently. No molecular cytogenetic information is available for most of these species. The crossability between 5 southern African Cucumis species (C. africanus, C. anguria, C. myriocarpus, C. zeyheri, and C. heptadactylus) has been reported; however, the evolutionary relationship among them is still unclear. Here, a molecular cytogenetic analysis using FISH with 5S and 45 S ribosomal DNA (rDNA) was used to investigate these Cucumis species based on sets of rDNA-bearing chromosomes (rch) types I, II and III. The molecular cytogenetic and phylogenetic results suggested that at least 2 steps of chromosomal rearrangements may have occurred during the evolution of tetraploid C. heptadactylus. In step 1, an additional 45 S rDNA site was observed in the chromosome (type III). In particular, C. myriocarpus had a variety of rch sets. Our results suggest that chromosomal rearrangements may have occurred in the 45 S rDNA sites. We propose that polyploid evolution occurred in step 2. This study provides insights into the chromosomal characteristics of African Cucumis species and contributes to the understanding of chromosomal evolution in this genus.

Retrotransposon- and microsatellite sequence-associated genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride.

Allopolyploidization is considered an essential evolutionary process in plants that could trigger genomic shock in allopolyploid genome through activation of transcription of retrotransposons, which may be important in plant evolution. Two retrotransposon-based markers, inter-retrotransposon amplified polymorphism and retrotransposon-microsatellite amplified polymorphism and a microsatellite-based marker, inter simple sequence repeat were employed to investigate genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride (2n = 4x = 38) which was derived from crossing between cultivated cucumber C. sativus L. (2n = 2x = 14) and its wild relative C. hystrix Chakr. (2n = 2x = 24). Extensive genomic changes were observed, most of which involved the loss of parental DNA fragments and gain of novel fragments in the allotetraploid. Among the 28 fragments examined, 24 were lost while four were novel, suggesting that DNA sequence elimination is a relatively frequent event during polyploidization in Cucumis. Interestingly, of the 24 lost fragments, 18 were of C. hystrix origin, four were C. sativus-specific, and the remaining two were shared by both species, implying that fragment loss may be correlated with haploid DNA content (genome size) of diploid parents. Most changes were observed in the first generation after polyploidization (S(1)) and stably inherited in the subsequent three generations (S(2)-S(4)), indicating that genomic changes might be a rapid driving force for the stabilization of allotetraploids. Sequence analysis of 11 of the 28 altered DNA fragments showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted into genome randomly and had a genome-wide effect on the allotetraploid evolution. Fluorescence in situ hybridization (FISH) analysis revealed a unique distribution of retrotransposon and/or microsatellite flanking sequences in mitotic and meiotic chromosomes, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.

Polyploidization facilitates biotechnological in vitro techniques in the genus Cucumis.

Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed.

Primary trisomics obtained from autotriploid by diploid reciprocal crosses in cucumber.

To promote cytogenetical studies on cucumber (Cucumis sativus L., 2n = 2x = 14), the reciprocal crosses were made between autotriploid and diploid for selecting the primary trisomics. Meanwhile, chromosome behavior during meiosis in autotriploid cucumber was investigated to look for cytological evidences for origin of primary trisomics. Many viable F(1) seeds were obtained from reciprocal crosses between autotriploid and diploid. The number of chromosomes of 56 surviving progenies varied from 14 to 28, with plants having 2n = 15 occurring at the highest frequency (51.8%). Primary trisomics were firstly obtained in this study. Four types of primary trisomics were isolated and they could be distinguished from each other, as well as diploid. Variable chromosome configurations, e.g. univalent, bivalents and trivalents were observed in many pollen mother cells of the autotriploid at metaphase I. Binomial chromosome distribution was observed at anaphase I and frequency of 8/13 was 6.25%. The meiosis of autotriploid, especially the class of gametes with eight chromosomes, gave the cytological evidence of producing 2x + 1 type gamete and could be induced into primary trisomic plants from progeny of autotriploid-diploid crosses. These studies have established a ground work for selecting a series of primary trisomics, and further using them for associating linkage groups with specific chromosomes in cucumber.

A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species.

Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.

Expression and sequence variation of the cucumber Por gene in the synthesized allotetraploid Cucumis x hytivus.

The molecular evolution of the cucumber Por gene in early generations of the synthesized allotetraploid Cucumis x hytivus was investigated. The results from gene expression analysis showed that the cucumber Por gene was silenced in the S(1) generation, and re-activated in the S(2) generation. In the S(3) and S(4) generations, the transcripts remained activated but sequence changes were observed. Further analysis indicated that base substitutions, including two transitions and one transversion, occurred in the S(1) and S(3) generation, respectively, and in the S(3) generation, an intron was found to be retained in the transcript. This indicates allopolyploidy induced rapid silencing and mutation of the cucumber Por gene. Further, gene mutations such as base substitution and intron retention are modes of evolution for duplicated genes in newly formed polyploids.

Changes of cytosine methylation induced by wide hybridization and allopolyploidy in Cucumis.

We previously demonstrated that allopolyploidization could induce phenotypic variations and genome changes in a newly synthesized allotetraploid in Cucumis. To explore the molecular involvement of epigenetic phenomena, we investigated cytosine methylation in Cucumis by using methylation-sensitive amplified polymorphism (MSAP). Results revealed a twofold difference in the level of cytosine methylation between the reciprocal F1 hybrids and the allotetraploid. Analysis of the methylation pattern indicated that methylation changed at 2.0% to 6.4% of total sites in both the F1 hybrids and the allotetraploid compared with their corresponding parents. Furthermore, 68.2% to 80.0% of the changed sites showed an increase in cytosine methylation and a majority of the methylated sites were from the maternal parent. Observations in different generations of the allotetraploid found that the extent of change in cytosine methylation pattern between the S1 and S2 was significantly higher than that between the S2 and S3, suggesting stability in advanced generations. Analysis of 7 altered sequences indicated their similarity to known functional genes or genes involved in regulating gene expression. Reverse transcription - polymerase chain reaction analysis suggested that at least two of the methylation changes might be related to gene expression changes, which further supports the hypothesis that DNA methylation plays a significant role in allopolyploidization.

Cytological diploidization and rapid genome changes of the newly synthesized allotetraploids Cucumis x hytivus.

We used a newly synthesized allotetraploid between C. sativus (2n = 2x = 14, n gametic chromosome number, x haploid chromosome number) and C. hystrix (2n = 2x = 24) to study the genomic events in its early generations. Results from cytological characterization of the F(1) and the allotetraploid progenies showed that the rate of bivalents in meiotic metaphase I of the F(1) was greatly improved by chromosome doubling, and further improved during the selfing process of allopolyploid resulting into relatively diploid-like meiosis. Extensive genomic changes were detected by amplified fragment length polymorphism analysis. The changes mainly involved loss of parental restriction fragments and gaining of novel fragments. The total detectable changes were from 11.1 to 32.1%, and the frequency of losing parental fragments was much higher than that of gaining novel fragments. Some of the changes were initiated as early as in the F(1) hybrid, whereas others occurred after chromosome doubling (polyploid formation). No significant differences were detected in the reciprocal F(1) hybrids and S(0) generations. But the data showed that the frequency of sequence losing in C. sativus was about two times higher than in the C. hystrix. Our results demonstrated that the sequence elimination was the major event of genomic changes, and it might provide the physical basis for the diploid-like meiotic behavior in the diploidization of the newly formed allopolyploids. Moreover, the results suggest that the sequence elimination was not caused by cytoplasmic factors, and might relate to genomic recombination and to the numbers of parental chromosome.

Molecular characterization of a Melon necrotic spot virus strain that overcomes the resistance in melon and nonhost plants.

Resistance of melon (Cucumis melo L.) to Melon necrotic spot virus (MNSV) is inherited as a single recessive gene, denoted nsv. No MNSV isolates described to date (e.g., MNSV-Malpha5), except for the MNSV-264 strain described here, are able to overcome the resistance conferred by nsv. Analysis of protoplasts of susceptible (Nsv/-) and resistant (nsv/nsv) melon cultivars inoculated with MNSV-264 or MNSV-Malpha5 indicated that the resistance trait conferred by this gene is expressed at the single-cell level. The nucleotide sequence of the MNSV-264 genome has a high nucleotide identity with the sequences of other MNSV isolates, with the exception of its genomic 3'-untranslated region (3'-UTR), where less than 50% of the nucleotides are shared between MNSV-264 and the other two MNSV isolates completely sequenced to date. Uncapped RNAs transcribed from a full-length MNSV-264 cDNA clone were infectious and caused symptoms indistinguishable from those caused by the parental viral RNA. This cDNA clone allowed generation of chimeric mutants between MNSV-264 and MNSV-Malpha5 through the exchange of the last 74 nucleotides of their coat protein (CP) open reading frames and the complete 3'-UTRs. Analysis of protoplasts of susceptible and resistant melon cultivars inoculated with chimeric mutants clearly showed that the MNSV avirulence determinant resides in the exchanged region. The carboxy-termini of the CP of both isolates are identical; therefore, the avirulence determinant likely consists of the RNA sequence itself. We also demonstrated that this genomic region contains the determinant for the unique ability of the isolate MNSV-264 to infect noncucurbit hosts (Nicotiana benthamiana and Gomphrena globosa).

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli.

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.