The percentage of DEGs involved in cellular processes and response to stimuli were higher for upregulated genes, whereas the percentage of DEGs involved in biological processes, localization, signaling, multicellular organismal and developmental processes were higher for downregulated genes. Similar relatively low percentages of DEGs were associated with metabolic and rhythmic processes and interspecies interaction. Additionally, a few upregulated genes were associated with immune system processes. In the molecular function class, the majority of DEGs were associated with binding and catalytic activities and these include up and down regulated genes. Those involved in structural molecule activity were upregulated, whereas those associated with molecular function regulator and transporter activities were downregulated . Finally, DEGs involved in molecular adaptor and molecular transducer activities were specifically upregulated. Together, these results suggest that the tomato defense response to TYLCV infection involved up and downregulated genes with similar molecular functions and biological processes. In order to validate the expression levels of these 12 selected DEGs were determined with RT-qPCR . Nine DEG were upregulated in line LA3473-R in response to TYLCV infection . Worldwide begomoviruses show a phylogeographic distribution, with most bipartite ones occurring in the New World , and most monopartite ones occurring in the Old World , and often in association with satellites DNAs that are either required for disease development or have no obvious effect on virulence . However, there are notable exception to this distribution, hydroponic rack system including the identification of indigenous NW monopartite begomoviruses infecting tomato in Peru, Ecuador and Northern Brazil .
The long distance spread and emergence of new begomoviruses has been mediated by the whitefly B. tabaci species Middle East Asia Minor 1 , which is a supervector of plant viruses . Furthermore, before the global spread of the whitefly supervector, begomovirus diseases were widely distributed in noncultivated plants in the NW and OW, with these viruses presumably spread by indigenous species of whiteflies . However, after the global spread of the polyphagous B. tabaci MEAM1 in the 1990s, indigenous begomoviruses were introduced into new cultivated and non-cultivated plant species . This has resulted in the emergence of similar diseases of crops and some weeds in different geographical regions. For example, the NW bipartite begomoviruses bean golden mosaic virus and bean golden yellow mosaic virus are different species that have independently evolved to cause beangolden mosaic disease of common bean in South America and North and Central America and the Caribbean Basin, respectively . Human activities have led to the long-distance intercontinental movement of numerous begomoviruses, which has further blurred the geographic separation of OW and NW begomoviruses, and accelerated the worldwide spread of NW bipartite economically important begomovirus diseases, e.g., introduction of NW bipartite squash leaf curl virus into the Middle East from the NW, and appearance of tomato leaf curl New Delhi virus in the Western Mediterranean Basin from the subcontinent of Asia . However, the most well documented and economically important example is the worldwide dissemination of the invasive OW monopartite begomovirus tomato yellow leaf curl virus . TYLCV was first introduced into the Dominican Republic during the early 1990s , and it has now invaded the Southern US, Mexico and the rest of the world, following the spread of the whitefly supervector .
Unfortunately, the tomato crop is highly permissive host for begomovirus infection, with ~90 tomato-infecting begomovirus species recognized by the International Committee on Taxonomy on Viruses . Tomato is one of the most consumed vegetables in the world . In Central America, tomato has become one of the most important crops in terms of area of cultivation and production . In Costa Rica , tomato is one of the most important vegetable crops, andis locally produced and sold as a fresh market crop often by small holder farmers. However, since the late 1980s, tomato production in CR has been impacted by different begomoviruses representing three examples of emergence and invasion: indigenous locally evolved, invasive from the region and exotic from a different continent. The indigenous tomato infecting begomovirus in CR is tomato yellow mottle virus , previously referred to as tomato geminivirus-Costa Rica . ToYMoV is a NW bipartite begomovirus associated with stunting and yellow mosaic/mottle of leaves . Growers first observed the tomato yellow mottle disease in the late 1980s , and ToYMoV was the predominant tomato-infecting begomovirus in CR until the introduction of the NW bipartite begomovirus tomato leaf curl Sinaloa virus in the late 1990s and the OW monopartite TYLCV in 2012 . Therefore, we consider ToYMoV to represent an indigenous, locally evolved begomovirus, ToLCSiV as an introduced virus from the region and TYLCV as an introduced virus from outside the region. Here, we utilized the tomato begomovirus situation in CR to examine their invasion biology, i.e., their interactions in terms of disease development, viral accumulation and viral genetics. To do this, we first completed the molecular and biological characterization of ToYMoV using full-length infectious DNA-A and DNA-B clones to fulfill fulfilling Koch’s postulates for the ToYMoD and to show that the virus primarily infected solanaceous species. Phylogenetic and sequence analyses indicated that ToYMoV may comprise a distinct lineage that is closely related to the squash leaf curl virus lineage of NW begomoviruses. This is consistent with the long period of local evolution in the region. Full-length infectious clones of an isolate of ToLCSiVfrom CR were generated and used to assess genetic interaction with ToYMoV.
The infectious clones of ToYMoV, ToLCSiV and TYLCV were then used to inoculate tomato plants with each virus clone and all combinations, and symptoms induced and viral DNA accumulation were determined. These findings are discussed in terms of the nature of the invasion biology of these viruses and to predict the future impact of these begomoviruses on tomato product in CR.The complete sequences of the cloned full-length DNA-A and DNA-B components of the GR1 isolate of ToYMoV collected in Grecia in 1990 and the L1 isolate of ToLCSiV collected in Liberia in 2002 were 2,574 nt and 2,547 nt , respectively, and 2,610 nt and 2,563 nt , respectively. The genome organization of these isolates is typical of NW bipartite begomoviruses, i.e., that is, a single gene on the virion -sense strand that encodes the CP, and four in the complementary -sense strand encoding the Rep, the transcriptional activator protein , the replication enhancer and the AC4 protein, respectively. Additionally, the CP and REn aa sequences of these isolates possess the N- and C-terminal motifs PWRlsAgT and AVRFATDr , respectively, which are characteristic of NW begomoviruses . The AC4 aa sequence contains the N-terminal myristoylation domain required for membrane targeting . The DNA-B components of these isolates have two ORFs, one in the v-sense strand encoding the nuclear shuttle protein , and one on the c-strand strand that encodes the movement protein . Pairwise sequence comparisons performed with SDT and the sequences of full-length DNA-A and DNA-B components of the GR1 isolate from Grecia revealed the highest identities with those of the DNA-A and DNA-B components of isolates of ToYMoV from CR . Consistent with these results, the ORFs of these components all had very high identities, i.e., all nt and aa sequence identities were ≥97%, with the exception of the AC4 aa sequence . Similar results were obtained for NTRs, including the common region and the hypervariable region of the DNA-B component . The next highest identities for the DNA-A component sequence were with NW bipartite begomoviruses from Latin America, including Sida chlorotic mottle virus from Brazil , tomato yellow leaf distortion virus from Cuba and tomato yellow vein streak virus from Chile . These results confirmed that the begomovirus infecting tomato plants with yellow mosaic/mottle symptoms in Grecia in 1990 was a variant of ToYMoV, which was named tomato yellow mottle virus-[CR:Grecia:1990] . The SDT analysis performed with the sequences of the complete DNA-A and DNA-B components of the L1 isolate from Liberia revealed the highest identities with those of the DNA-A and DNA-B components of isolates of ToLCSiV from CR and NI , cannabis vertical grow system whereas identities were lower with available partial sequences of isolates from MX. Similar results were obtained in comparisons made with nt and aa sequences of the ORFs , whereas common regionidentities ranged from ≥93 to 97% and identities for HVR of the DNA-B component ranged from ≥95 to 98% .
The next highest identities for the sequences of the DNAA component were with NW bipartite begomoviruses from Latin America, including Sida interveinal bright yellow virus from MX , Sida yellow vein virus from Honduras and chino del tomate virus from MX . The ToYMoV common region sequence contains all the cis-regulatory elements implicated in virus replication and gene expression, e.g., the conserved geminivirus stemloop structure with the nonanucleotide sequence TAATATT↓AC, the Rep high-affinity binding site and the canonical AC1 TATA box and G-box . The Rep high-affinity binding site is composed of two direct repeats of GGTGT adjacent to the AC1 TATA-box, and an upstream inverted repeat ACACC . The Rep iteron-related domain is MPPPKKFRLS , which is predicted to interact with the core iteron sequence GGTGT . Here, it is worth nothing that it has been proposed that ToYMoV may possess a unique 8 nt iteron sequence that is found in all members of the SLCuV lineage . The DNA-A and DNA-B components of ToLCSiV share a common region of 174 nt, which is 96% identical, indicating these are cognate components. The Rep high affinity binding site of ToLCSiV consists of two direct repeats of the GGGGT adjacent to the AC1 TATA-box, and one inverted ACTCC motif . The Rep IRD is MPSVKRFKVS , which is predicted to recognize the GGGGT iteron .In the phylogenetic tree generated with the sequences of the complete DNA-A components, the ToYMoV isolates from CR were placed together in a strongly supported clade , consistent with the low level of sequence divergence among these isolates collected ~22 years apart . This clade was erected as a sister group of the SLCuV lineage, which is composed primarily of cucurbit-infecting begomoviruses from the Southern US, MX and Central America. In the phylogenetic analysis performed with the complete DNA-B sequences, ToYMoV was also placed as a sister clade of the SLCuV lineage . Interestingly, in this DNA-B tree, the ToYMoV isolates were most closely related with bean leaf crumple virus from Colombia . The ToLCSiV isolates from CR form a strongly supported clade with the isolate from NI . This clade was part of the AbMV lineage, which includes crop- and weed-infecting begomoviruses from North and Central America and the Caribbean Basin, such as chino del tomate virus from MX and ToMoV from Florida . In the tree generated with the complete DNA-B sequences, the ToLCSiVisolates from CR and NI were also placed together in a strongly supported clade in the AbMV lineage .RDP4 analysis reveal a single recombination event in the DNA-A component of all four ToYMoV isolates , whereas no recombination was detected in the DNAB component nor in the DNA-A and DNA-B components of ToLCSiV. The recombination event in the ToYMoV DNA-A was 424 nt and spans nts 2056 to 2479 and includes the 5’ end of the AC1 ORF, the entire AC4 ORF and the 5´ sequence of the common region. Thus, this event was in the well-known begomovirus recombination hot-spot region . The RDP4 analysis further indicated that the recombinant region was derived from an uncharacterized minor parent, whereas the major parent was most similar to tomato chlorotic leaf distortion virus from Venezuela .The infectivity and pathogenicity of the full-length cloned DNA-A and DNA-B components of ToYMoV-[CR:Gre:90] were established by particle bombardment inoculation of N. benthamiana and tomato seedlings . By 14 dpb, all of the bombarded N. benthamiana plants were stunted and newly emerged leaves showed epinasty, crumpling, yellow mosaic/mottle and vein yellowing ; whereas tomato seedlings were stunted and newly emerged leaves had developed epinasty, crumpling and mild yellow mosaic/mottle by 14dpb . In N. benthamiana and tomato plants, symptoms in ToYMoV-infected plants became progressively milder by 21 dpb . Notably, symptoms in tomato plants following bombardment of the infectious ToYMoV clones were similar to those of the ToYMoD of tomato in the field in CR, thereby fulfilling Koch´s postulates for this disease.