Genetic variability and evolutionary dynamics of atypical Papaya ringspot virus infecting Papaya.

Papaya ringspot virus biotype-P is a detrimental pathogen of economically important papaya and cucurbits worldwide. The mutation prone feature of this virus perhaps accounts for its geographical dissemination. In this study, investigations of the atypical PRSV-P strain was conducted based on phylogenetic, recombination and genetic differentiation analyses considering of it's likely spread across India and Bangladesh. Full length genomic sequences of 38 PRSV isolates and 35 CP gene sequences were subjected to recombination analysis. A total of 61 recombination events were detected in aligned complete PRSV genome sequences. 3 events were detected in complete genome of PRSV strain PK whereas one was in its CP gene sequence. The PRSV-PK appeared to be recombinant of a major parent from Bangladesh. However, the genetic differentiation based on full length genomic sequences revealed less frequent gene flow between virus PRSV-PK and the population from America, India, Colombia, other Asian Countries and Australia. Whereas, frequent gene flow exists between Pakistan and Bangladesh virus populations. These results provided evidence correlating geographical position and genetic distances. We speculate that the genetic variations and evolutionary dynamics of this virus may challenge the resistance developed in papaya against PRSV and give rise to virus lineage because of its atypical emergence where geographic spread is already occurring.

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions.

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf patches radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in mixed-infected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90-120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

Mutualistic and antagonistic interactions differ in wild and domesticated papaya (Carica papaya) in its centre of origin.

Shifts in phenotypes derived from the domestication syndromes impact plant performance but may also affect interactions with other species in the community (e.g. mutualists and antagonists). Moreover, plantations often differ from the natural conditions experienced by the wild relatives of cultivated plants, potentially altering the nature of ecological interactions. However, apart from herbivory, little is known about how domestication and cultivation practices (e.g. insecticide application) can modify multiple ecological interactions simultaneously in wild and domesticated plants. In four sites on the Yucatan Peninsula, we compared the diversity of mutualists (e.g. moths) and antagonists (e.g. viruses) in wild and domesticated plants of papaya. For each individual, we recorded floral visitors and rates of visitation at three time periods during the day. We recorded type and percentage of damage by antagonists in three leaves of all individuals. Finally, we explored if plant sex had an effect on the interaction with floral visitors. The main floral visitors were ants and Trigona species, whereas viruses caused the main type of foliar damage. Wild individuals had a higher diversity and visitation rate of floral visitors, and less foliar damage from antagonists. Wild male individuals were more visited, but we observed a similar amount and diversity of damage in both sexes. The time of day did not have an effect on diversity of floral visitors. Together, cultivation practices and domestication appear to have an effect on the reduction in diversity of floral visitors in domesticated papaya, as well as an increase in foliar damage.

Transmission of the Bean-Associated Cytorhabdovirus by the Whitefly Bemisia tabaci MEAM1.

The knowledge of genomic data of new plant viruses is increasing exponentially; however, some aspects of their biology, such as vectors and host range, remain mostly unknown. This information is crucial for the understanding of virus-plant interactions, control strategies, and mechanisms to prevent outbreaks. Typically, rhabdoviruses infect monocot and dicot plants and are vectored in nature by hemipteran sap-sucking insects, including aphids, leafhoppers, and planthoppers. However, several strains of a potentially whitefly-transmitted virus, papaya cytorhabdovirus, were recently described: (i) bean-associated cytorhabdovirus (BaCV) in Brazil, (ii) papaya virus E (PpVE) in Ecuador, and (iii) citrus-associated rhabdovirus (CiaRV) in China. Here, we examine the potential of the Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) to transmit BaCV, its morphological and cytopathological characteristics, and assess the incidence of BaCV across bean producing areas in Brazil. Our results show that BaCV is efficiently transmitted, in experimental conditions, by B. tabaci MEAM1 to bean cultivars, and with lower efficiency to cowpea and soybean. Moreover, we detected BaCV RNA in viruliferous whiteflies but we were unable to visualize viral particles or viroplasm in the whitefly tissues. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. BaCV was detected in five out of the seven states in Brazil included in our study, suggesting that it is widely distributed throughout bean producing areas in the country. This is the first report of a whitefly-transmitted rhabdovirus.

Genomic variation between PRSV resistant transgenic SunUp and its progenitor cultivar Sunset.

BACKGROUND: The safety of genetically transformed plants remains a subject of scrutiny. Genomic variants in PRSV resistant transgenic papaya will provide evidence to rationally address such concerns. RESULTS: In this study, a total of more than 74 million Illumina reads for progenitor 'Sunset' were mapped onto transgenic papaya 'SunUp' reference genome. 310,364 single nucleotide polymorphisms (SNPs) and 34,071 small Inserts/deletions (InDels) were detected between 'Sunset' and 'SunUp'. Those variations have an uneven distribution across nine chromosomes in papaya. Only 0.27% of mutations were predicted to be high-impact mutations. ATP-related categories were highly enriched among these high-impact genes. The SNP mutation rate was about 8.4 × 10- 4 per site, comparable with the rate induced by spontaneous mutation over numerous generations. The transition-to-transversion ratio was 1.439 and the predominant mutations were C/G to T/A transitions. A total of 3430 nuclear plastid DNA (NUPT) and 2764 nuclear mitochondrial DNA (NUMT) junction sites have been found in 'SunUp', which is proportionally higher than the predicted total NUPT and NUMT junction sites in 'Sunset' (3346 and 2745, respectively). Among all nuclear organelle DNA (norgDNA) junction sites, 96% of junction sites were shared by 'SunUp' and 'Sunset'. The average identity between 'SunUp' specific norgDNA and corresponding organelle genomes was higher than that of norgDNA shared by 'SunUp' and 'Sunset'. Six 'SunUp' organelle-like borders of transgenic insertions were nearly identical to corresponding sequences in organelle genomes (98.18 ~ 100%). None of the paired-end spans of mapped 'Sunset' reads were elongated by any 'SunUp' transformation plasmid derived inserts. Significant amounts of DNA were transferred from organelles to the nuclear genome during bombardment, including the six flanking sequences of the three transgenic insertions. CONCLUSIONS: Comparative whole-genome analyses between 'SunUp' and 'Sunset' provide a reliable estimate of genome-wide variations and evidence of organelle-to-nucleus transfer of DNA associated with biolistic transformation.

Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite.

Codiaeum variegatum (common name, garden croton) is an ornamental plant grown for its bright yellow variegated leaf morphology. Two C. variegatum plants with upward leaf curling and vein swelling symptoms were collected in Faisalabad, Pakistan. Sequencing of clones obtained by PCR amplification with specific primers showed one plant infected with the monopartite begomoviruses pedilanthus leaf curl virus (PeLCV) and papaya leaf curl virus (PaLCuV) and the other to be infected with only PeLCV. Both plants also harboured a betasatellite that was distinct from all previously identified betasatellites, for which the name "codiaeum leaf curl betasatellite" (CoLCuB) is proposed. This is the first identification of a begomovirus and an associated betasatellite infecting C. variegatum in Pakistan. Both PeLCV and PaLCuV cause problems in a number of crop plants, and C. variegatum may act as a reservoir for these agriculturally important viruses. The precise impact and geographical distribution of the newly identified CoLCuB will be investigated.

Metaviromic analysis reveals coinfection of papaya in western Kenya with a unique strain of Moroccan watermelon mosaic virus and a novel member of the family Alphaflexiviridae.

Severe mottling symptoms were observed on Carica papaya L. in Koyonzo, Kakamega County, Kenya. Total RNA was sequenced via an RNAtag-seq workflow. Assembled contigs indicated the presence of a divergent strain of Moroccan watermelon mosaic virus (genus Potyvirus) with a complete genome length of 9,733 nt (GenBank accession no. MN418119). Additionally, the complete genome sequence of a novel member of the viral genus Allexivirus was determined (GenBank accession no. MN418120). The genome contains six open reading frames (ORFs) that show varying degrees of sequence similarity to members of the genus Allexivirus; however, it appears to lack an ORF encoding a nucleic-acid-binding homolog. The tentative name "papaya virus A" (PaVA) has been proposed for this virus.

A multiplex RT-PCR method to detect papaya meleira virus complex in adult pre-flowering plants.

Papaya sticky disease (PSD), which can destroy orchards, was first attributed to papaya meleira virus (PMeV). However, the discovery of papaya meleira virus 2 (PMeV2) associated with PSD plants impose the need to detect this viral complex. We developed a multiplex RT-PCR (mPCR) technique capable of detecting two viruses in a single assay from pre-flowering plant samples, which is a useful tool for early diagnosis of PSD. We also determined the limit of detection (LOD) using asymmetric plasmid dilutions of both PMeV and PMeV2, which revealed that a higher titer of one virus prevents detection of the other. Thus, this technique is an alternative method for detecting PMeV and PMeV2 in a single reaction.

Differential Accumulation of Innate- and Adaptive-Immune-Response-Derived Transcripts during Antagonism between Papaya Ringspot Virus and Papaya Mosaic Virus.

Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved.

A Double Mutation in the Conserved Motifs of the Helper Component Protease of Papaya Leaf Distortion Mosaic Virus for the Generation of a Cross-Protective Attenuated Strain.

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.

DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya.

Papaya ringspot virus (PRSV) infections in papaya result in heavy yield losses, severely affecting the papaya industry worldwide, and hence warranting for effective control measures. In the past, transgenic papaya cultivars were developed that overexpressed parts of the PRSV genome and exhibited high levels of virus resistance. In the present study, a non-transgenic approach was employed, in which in vitro produced dsRNA molecules derived from a PRSV isolate from South India (PRSV-Tirupati) was tested for dsRNA-mediated protection against two isolates of PRSV through topical application of the dsRNA on papaya. The results showed that the dsRNA molecules from both the coat protein (CP) and helper component-proteinase (HC-Pro) genes of the PRSV-Tirupati isolate conferred 100 % resistance against PRSV-Tirupati infection. Further, the same dsRNA molecules were highly effective against the PRSV-Delhi isolate on the papaya cv. Pusa Nanha, conferring a resistance of 94 % and 81 %, respectively. Systemic papaya leaves of the dsRNA-treated plants were virus-free at 14 days post-inoculation, confirming the robustness of this non-transgenic virus control strategy. In contrast, the control TMV dsRNA did not protect against the PRSV infection. This study on the topical application of dsRNA opened up a new avenue for the control of papaya ringspot disease worldwide.

Papaya Ringspot Virus Isolates From Papaya in Bangladesh: Detection, Characterization, and Distribution.

Papaya ringspot virus (PRSV) is the major constraint to papaya (Carica papaya) production in Bangladesh. Disease symptoms occurred in 90 to 100% of the plants surveyed. Full-length genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse-transcription PCR(RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74 and 83% identical to each other, respectively, at the nucleotide and amino acid levels. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical, respectively, to other full-length PRSV sequences at the nucleotide level. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a branch distinct from the other PRSV sequences based on nucleotide and amino acid sequence comparisons. Comparisons of the genome sequences of these two strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment highly identical to PRSV-Meghalaya (MF356497). The other recombinant event contained a 5,105-nucleotide fragment highly identical to PRSV-China (KY933061). The occurrence rates of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh were approximately 19 and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The full-length genome sequences of these new PRSV strains and their distribution provide important information regarding the dynamics of papaya ringspot virus infections in papaya in Bangladesh.

Sequencing, genome analysis and prevalence of a cytorhabdovirus discovered in Carica papaya.

The complete genome of a new rhabdovirus infecting papaya (Carica papaya L.) in Ecuador, named papaya virus E, was sequenced and characterized. The negative-sense single-stranded RNA genome consists of 13,469 nucleotides with six canonical open reading frames (ORFs) and two accessory short ORFs predicted between ORFs corresponding to P3 (movement protein) and M (matrix protein). Phylogenetic analyses using amino acid sequences from the nucleocapsid, glycoprotein and polymerase, grouped the virus with members of the genus Cytorhabdovirus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives. The 3' leader and 5' trailer sequences were 144 and 167 nt long, respectively, containing partially complementary motifs. The motif 3'-AUUCUUUUUG-5', conserved across rhabdoviruses, was identified in all but one intergenic regions; whereas the motif 3'-ACAAAAACACA-5' was found in three intergenic junctions. This is the first complete genome sequence of a cytorhabdovirus infecting papaya. The virus was prevalent in commercial plantings of Los Ríos, the most important papaya producing province of Ecuador. Recently, the genome sequence of bean-associated cytorhabdovirus was reported. The genome is 97% identical to that of papaya virus E, indicating that both should be considered strains of the same virus.

Empoasca papayae (Hemiptera: Cicadellidae)-Mediated Transmission of Papaya Meleira Virus-Mexican Variant in Mexico.

Papaya meleira virus (PMeV) causes sticky disease in Carica papaya in Brazil and Mexico. Despite its economic importance and the need for effective phytosanitary control, it remains unknown whether any insect is the vector of this virus. The aim of this work was to identify potential insect vectors of the PMeV-Mexican variant (PMeV-Mx) and determine whether these potential vectors are capable of transmitting the virus. Adult insects were collected in papaya fields in the south-southeast region of Mexico and were identified morphologically and molecularly. Their abundance and frequency were determined, and quantitative reverse transcription polymerase chain reaction was performed to establish if they carried PMeV-Mx. The Cicadellidae family (Hemiptera) was the most diverse and abundant, and Empoasca papayae was the most abundant species and had the highest virus titers. PMeV-Mx transmission assays were conducted under controlled conditions using E. papayae on C. papaya 'Maradol'. E. papayae was a carrier of PMeV-Mx at 6 h after exposure, and its viral titer increased with time, peaking at 2.125 pg/µl of PMeV-Mx RNA from 20 ng/µl of cDNA, 5 days after exposure (dae). From 14 days after plants were exposed to insects, PMeV-Mx was detected and quantified in 100% of the evaluated papaya plants, whose viral RNA titer increased from 0.06 (21 dae) to 26.6 pg/µl of PMeV-Mx RNA (60 dae) from 20 ng/µl of cDNA. Three months later, these plants developed sticky disease symptoms, demonstrating that E. papayae is capable of transmitting PMeV-Mx to C. papaya 'Maradol'.

Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization.

Forty-five papaya samples showing severe leaf curl symptoms were tested by PCR with a degenerate primer set for virus species in the genus Begomovirus. Of these, 29 were positive for tomato leaf curl Bangladesh virus (ToLCBV). The complete genome sequences of ToLCBV (GenBank accession no. MH380003) and its associated tomato leaf curl betasatellite (ToLCB) (MH397223) from papaya isolate Gaz17-Pap were determined and characterized. Defective betasatellites were found in ToLCBV-positive papaya isolates Gaz19-Pap, Gaz20-Pap and Gaz21-Pap. This study confirmed that papaya is a host of ToLCBV, ToLCB, and other defective and recombinant DNA satellites in Bangladesh.

Ultra-sensitive detection of papaya ringspot virus using single-tube nested PCR.

Aphid-transmitted papaya ringspot virus (PRSV) is the greatest disease threat to the commercial production of papaya worldwide. Specific ultrasensitive assays are important for the early detection of PRSV in the field. We have developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of PRSV. The annealing temperatures and concentrations of both primer pairs were optimized to reduce potential competition between primer sets in STNP. The assay is more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. STNP is capable of detecting PRSV in plants 7 days post-inoculation, whereas RT-PCR and ELISA are capable of detecting PRSV 14 to 21 days post-inoculation. This new assay can also detect PRSV from virus infected but asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV infection rates in the field and in imported plant samples. Keywords: early detection; quarantine protocols.

Profile of siRNAs derived from green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus in infected papaya plants.

We used green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus (PLDMV-GFP) to track PLDMV infection by fluorescence. The virus-derived small interfering RNAs (vsiRNAs) of PLDMV-GFP were characterized from papaya plants by next-generation sequencing. The foreign GFP gene inserted into the PLDMV genome was also processed as a viral gene into siRNAs by components involved in RNA silencing. The siRNAs derived from PLDMV-GFP accumulated preferentially as 21- and 22-nucleotide (nt) lengths, and most of the 5'-terminal ends were biased towards uridine (U) and adenosine (A). The single-nucleotide resolution map revealed that vsiRNAs were heterogeneously distributed throughout the PLDMV-GFP genome, and vsiRNAs derived from the sense strand were more abundant than those from the antisense strand. The hotspots were mainly distributed in the P1 and GFP coding region of the antisense strand. In addition, 979 papaya genes targeted by the most abundant 1000 PLDMV-GFP vsiRNAs were predicted and annotated using GO and KEGG classification. Results suggest that vsiRNAs play key roles in PLDMV-papaya interactions. These data on the characterization of PLDMV-GFP vsiRNAs will help to provide insight into the function of vsiRNAs and their host target regulation patterns.

Two agroinfection-compatible fluorescent protein-tagged infectious cDNA clones of papaya leaf distortion mosaic virus facilitate the tracking of virus infection.

Papaya leaf distortion mosaic virus (PLDMV, the genus Potyvirus) is an emerging threat to papaya production. Here, agroinfection-compatible fluorescent protein-tagged PLDMV infectious cDNA clones driven by the Cauliflower mosaic virus 35S promoter were successfully constructed using one-step Gibson assembly. The clones were directly transformed into Agrobacterium tumefaciens to prevent potential problems such as plasmid instability during propagation in Escherichia coli. Ninety-five percent of papaya seedlings infected with PLDMV-GFP or PLDMV-mCherry developed systemic symptoms typical of those caused by wild-type PLDMV. Green and mCherry red fluorescence was observed in leaves, stems, and roots of infected papaya plants. The fluorescent protein-tagged agroinfectious PLDMV cDNA clones were stable in papaya for more than 90 days and during six serial passages at 30-day intervals. The availability of these infectious clones will contribute to research on PLDMV-host interactions and can be applied in the papaya breeding program for PLDMV resistance.

Characterization of Papaya ringspot virus isolates infecting transgenic papaya 'Huanong No.1' in South China.

In 2006, the release and cultivation of the genetically modified papaya cultivar 'Huanong No.1' successfully controlled the destructive papaya ringspot disease caused by Papaya ringspot virus (PRSV) in South China. However, some transgenic papaya plants from Guangdong and Hainan are found infected by PRSV. In this study, Field investigation was carried out and susceptible transgenic papaya samples were collected during 2012-2016. Twenty representative isolates were artificially inoculated into Cucurbita pepo and commercialised 'Huanong No.1' papaya, and results indicated that the plants showed obvious disease symptoms. Phylogenetic analysis of CP genes of 120 PRSV-infected isolates showed that PRSV can be divided into three groups. Isolates from Guangdong and Hainan belong to Group III, which is further divided into two subgroups. The isolates collected in this study have greatly diverged from the previously reported dominant strains Ys, Vb and Sm in South China, indicating that they belong to a new lineage. Further analysis showed a highly genetic differentiation between isolates, and 27.1% of the isolates were identified as recombinants on the basis of CP nucleotide sequences. These results indicate that the genetic variation of PRSV and the formation of the new virus lineage may explain the loss of transgenic papaya resistance in South China.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.