Cucurbit aphid-borne yellows virus (CABYV) is a positive-sense single stranded RNA virus belonging to the Polerovirus genus [1] of the Solemoviridae family. In Brazil, the occurrence of CABYV has been reported in passion fruit plants showing symptoms such as blisters, mosaic and leaf deformation [2]. Later, a totally different type of CABYV isolate infecting melon plants, showing severe yellowing disease (called “Amarelão do meloeiro” in Brazil), was reported [3], since this isolate was recombinant virus in 3' region including coat protein (CP) and minor CP genes (i.e., P3/P5 region). The distribution of this recombinant isolate expands from Ceará to Bahia states in Brazil, showing the establishment of this recombinant strain in melon fields widely in Brazil. In addition, this virus was very peculiar polerovirus, hence its transmissibility by whitefly was reported later [4]. Thus, the name of “cucurbit whitefly-borne yellows virus” (CWBYV) was suggested for this recombinant isolate [4].
To date, there are no efficient management strategies available against this disease. There are still no melon resistant varieties to this recombinant strain of CABYV. To develop the resistant or immune variety, the biological characterization of the virus and virus challenge trials to test plants are essential, however, our trials of mechanical inoculation of CABYV to host and test plants failed [3]. Thus, the full-length genomic cDNA of this CABYV recombinant strain was constructed in this study to have inoculum source and for reverse genetic study.
The CABYV M3 isolate, collected in Icapuí county, Ceará state, Brazil [3] was used to prepare cDNA fragments of the genome. The total RNA, from the infected leaves stored at freezer –70 ˚C, was extracted using silica resin-based protocol according to [5]. The cDNA of the complete genome of the M3 isolate was amplified dividing into two fragments, the 5' and 3' regions with an overlapping region of 20 nts, by RT-PCR. At first, cDNA was synthesized with SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Waltham, USA) using the specific primer which anneals the genomic 3’ end (CABYV_Infectious_pJLRz_Rev: 5' GGA GAT GCC ATG CCG ACC CAC ACC GAA ACG CCA GGG 3') (Fig. 1a). However, the consequence PCR amplification with primer pair (CABYV_Infectious pJLRz_Rev and CABYV_3325_For: 5' TCC ATT CTC AAT GAA TTG CGG TC 3') was not successful in many trials. With the hypothesis that the highly stable secondary structure in the 3’ end region of the genome disturbs the PCR, the cDNA synthesis method was changed. Before reverse transcription reaction, the poly(A) tail was added tothe extracted RNA using poly(A) polymerase (New England Biolabs, Ipswich, USA), then cDNA was synthesized using oligo dT50 primer [3]. With this cDNA, almost half of the genome from the 3’ end was amplified using LongAmp Taq DNA polymerase (New England Biolabs) and specific primers (CABYV_Infectious pJLRz_Rev and CABYV_3325_For). To amplify the 5' region of the viral genome, cDNA was synthesized with random primers using SuperScript IV reverse transcriptase and PCR was performed using CABYV_Infectious_35s_For (5' GTT CAT TTC ATT TGG AGA GGA CAA AAG ATA CGA GCG GGT G 3') and CABYV_3348_Rev (5' AGA CCG CAA TTC ATT GAG AAT G 3') primers. These cDNA fragments were cloned into pCR4 TOPO plasmid vector (Thermo Fisher Scientific) in Escherichia coli DH10B strain (Thermo Fisher Scientific) and cultured at 28 ˚C for up to two days. The plasmids were purified using Wizard Plus SV Minipreps DNA Purification Systems (Promega, Madison, USA) and sequenced by Sanger method for the insert cDNA confirmation. The selected clones were re-amplified by PCR to join the two fragments into one using “Gibson Assembly” protocol [6]. The binary vector backbone of pJL89, containing dual promoter of cauliflower mosaic virus 35S and hepatitis delta virus ribozyme prior to nopaline synthase terminator, was linearized by PCR using primer pair of pJL89_Gibson_rev (5' CCT CTC CAA ATG AAA TGA ACT TCC 3') and pJL89_Gibson_for (5' GGG TCG GCA TGG CAT CTC 3'). These three fragments share the overlapping sequences of 20 nts in both 5’ and 3’ ends to join with Gibson Assembly (Fig. 1a). Again, the insert of the selected two plasmids were totally sequenced by Sanger method. Agrobacterium tumefaciens strain GV3101:pMP90 was transformed with the CABYV-M3 constructs.
For the first trial to evaluate the infectivity of CABYV-M3 clones, two CABYV clones in A. tumefaciens cells named CABYV-M3a and CABYV-M3b, were inoculated according to [6] in five plants of Cucumis melo (cv. Goldex, which is a susceptible variety for this virus) for each clone. As a control, A. tumefaciens containing pJL89 vector without insert was agro-infiltrated. After 7 to 10 days of inoculation, agroinfiltrated plants with the CABYV-M3a and M3b clones showed symptoms of interveinal chlorosis in the inoculated leaves. At 30 days post-infiltration (dpi), melon leaves showed strong interveinal chlorosis (Fig. 1b). Control plants were asymptomatic (Fig. 1b). The upper leaves (not-infiltrated) from five inoculated plants with CABYV-M3a and CABYV-M3b clones were prepared as pooled samples separately for RNA extraction and RT-PCR detection using specific primers, CABYV_2663 For (5' AAC GCC CTC GGA ATT GAT CAC C 3') and CABYV_3728 Rev (5' TTG CTG AAT ACA AAT GCT TGC ATC 3'). The amplified cDNA fragments of expected size (1066 bps) in both samples were confirmed by agarose gel electrophoresis (Fig.1c). The amplicon was sequenced by Sanger method and the infection by the M3a and M3b clones was confirmed.
Besides, the inoculation of CABYV-M3a, to test plants for host response study, was performed in two independent trials. The following plants were used in this host response study: two cultivars of squash (Cucurbita maxima × C. moschata cv. Tetsukabuto and C. maxima cv. Moranga Coroa), cucumber (Cucumis sativus cv. Caipira 201), watermelon (Citrullus lanatus cv. Crimson Sweet), zucchini (Curcubita pepo cv. Caserta), West Indian gherkin (Cucumis anguria cv. Isla 163) and melon (C. melo cv. Calábria). The melon cv. Goldex (susceptible control) were inoculated again. At 15 days after agro-infiltration, the upper leaves (not agroinfiltrated) were collected for CABYV-M3a detection by RT-PCR using primers CABYV_2663 For and CABYV_3728 Rev.
Of six cucurbit species tested in the host response experiment, the CABYV-M3a was detected in three: squash (hybrid of C. maxima × C. moschata, 7 positive/8 inoculated plants and C. maxima cv. Moranga Coroa, 6/8), West Indian gherkin (Cucumis anguria, 6/6), and melon (C. melo cv. Calábria, 10/12 and cv. Goldex, 12/12) (Table 1). CABYV-M3a infection was not confirmed in cucumber (C. sativus), zucchini (C. pepo) and watermelon (C. lanatus) by RT-PCR nor symptom appearance. Symptoms observed in squash and West Indian gherkin were leaf edge chlorosis from 30 dpi. In melon plants (cvs. Goldex and Calábria), the symptom was severe throughout the plant, as observed in infected melon plants in the fields [3].
In previous report of whitefly transmission test, both squash and West Indian gherkin were susceptible to the field collected M1 isolate of CABYV [4], therefore, the susceptibility of these plant species to CABYV infectious clone corroborates our former study. Again, watermelon was not susceptible for the infectious clone as well as former study [4], which may explain the absence of this virus occurrence in watermelon fields often located near by the melon plantation.
In conclusion, the full-length genomic cDNA clones of CABYV produced using the Gibson Assembly procedure are infectious. This clone will be an important tool for future study in developing the resistant melon varieties to CABYV infection.