Utilizing microarray spot characteristics to improve cross-species hybridization results

AbstractCross-species hybridization (CSH), i.e., the hybridization of a (target) species RNA to a DNA microarray that represents another (reference) species, is often used to study species diversity. However, filtration of CSH data has to be applied to extract valid information. We present a novel approach to filtering the CSH data, which utilizes spot characteristics (SCs) of image-quantification data from scanned spotted cDNA microarrays. Five SCs that were affected by sequence similarity between probe and target sequences were identified (designated as BS-SCs). Filtration by all five BS-SC thresholds demonstrated improved clustering for two of the three examined experiments, suggesting that BS-SCs may serve for filtration of data obtained by CSH, to improve the validity of the results. This CSH data-filtration approach could become a promising tool for studying a variety of species, especially when no genomic information is available for the target species

Parasitization by the wasp Eretmocerus mundus induces transcription of genes related to immune response and symbiotic bacteria proliferation in the whitefly Bemisia tabaci

<p>Abstract</p> <p>Background</p> <p>The whitefly <it>Bemisia tabaci </it>(Gennadius) (Hemiptera: Aleyrodidae), and the viruses it transmits, are a major constraint to growing vegetable crops worldwide. Although the whitefly is often controlled using chemical pesticides, biological control agents constitute an important component in integrated pest management programs, especially in protected agriculture. One of these agents is the wasp <it>Eretmocerus mundus </it>(Mercet) (Hymenoptera: Aphelinidae)<it>. E. mundus </it>lays its egg on the leaf underneath the second-third instar nymph of <it>B. tabaci</it>. First instars of the wasp hatch and penetrate the whitefly nymphs. Initiation of parasitization induces the host to form a capsule composed of epidermal cells around the parasitoid. The physiological and molecular processes underlying <it>B. tabaci-E. mundus </it>interactions have never been investigated.</p> <p>Results</p> <p>We used a cDNA microarray containing 6,000 expressed sequence tags (ESTs) from the whitefly genome to study the parasitoid-whitefly interaction. We compared RNA samples collected at two time points of the parasitization process: when the parasitoid first instar starts the penetration process and once it has fully penetrated the host. The results clearly indicated that genes known to be part of the defense pathways described in other insects are also involved in the response of <it>B. tabaci </it>to parasitization by <it>E. mundus</it>. Some of these responses included repression of a serine protease inhibitor (<it>serpin</it>) and induction of a melanization cascade. A second set of genes that responded strongly to parasitization were bacterial, encoded by whitefly symbionts. Quantitative real-time PCR and FISH analyses showed that proliferation of <it>Rickettsia</it>, a facultative secondary symbiont, is strongly induced upon initiation of the parasitization process, a result that supported previous reports suggesting that endosymbionts might be involved in the insect host's resistance to various environmental stresses.</p> <p>Conclusion</p> <p>This is the first study to examine the transcriptional response of a hemipteran insect to attack by a biological control agent (hymenopterous parasitoid), using a new genomic approach developed for this insect pest. The defense response in <it>B. tabaci </it>involves genes related to the immune response as described in model organisms such as <it>Drosophila melanogaster</it>. Moreover, endosymbionts of <it>B. tabaci </it>appear to play a role in the response to parasitization, as supported by previously published results from aphids.</p

Derivation of species-specific hybridization-like knowledge out of cross-species hybridization results

BACKGROUND: One of the approaches for conducting genomics research in organisms without extant microarray platforms is to profile their expression patterns by using Cross-Species Hybridization (CSH). Several different studies using spotted microarray and CSH produced contradicting conclusions in the ability of CSH to reflect biological processes described by species-specific hybridization (SSH). RESULTS: We used a tomato-spotted cDNA microarray to examine the ability of CSH to reflect SSH data. Potato RNA was hybridized to spotted cDNA tomato and potato microarrays to generate CSH and SSH data, respectively. Difficulties arose in obtaining transcriptomic data from CSH that reflected those obtained from SSH. Nevertheless, once the data was filtered for those corresponding to matching probe sets, by restricting proper cutoffs of probe homology, the CSH transcriptome data showed improved reflection of those of the SSH. CONCLUSIONS: This study evaluated the relative performance of CSH compared to SSH, and proposes methods to ensure that CSH closely reflects the biological process analyzed by SSH

Tomato yellow leaf curl virus: A whitefly-transmitted geminivirus with a single genomic component

The genome of the tomato yellow leaf curl virus (TYLCV), a Bemisia tabaci-transmitted geminivirus, was cloned. All clones obtained were of one genomic molecule, analogous to DNA A of African cassava mosaic virus. Nucleotide sequence analysis of the TYLCV genome showed that it comprises 2787 nucleotides, encoding six open reading frames, two on the virion strand and four on the complementary strand. All of them have counterparts in other geminiviruses. Dimeric copies of the cloned viral genome were introduced into tomato plants by agroinoculation. Severe yellow leaf curl disease symptoms developed in all of them. Effective whitefly-mediated transmission of the virus from agroinoculated plants to test plants demonstrated that the cloned molecule carries all the information needed for virus replication, systemic infection, and transfer by whiteflies. Restriction and hybridization analyses of viral DNA forms in infected plants and viruliferous whiteflies did not support the presupposed existence of a second genomic component. This is the first report of a whitefly-transmitted geminivirus that possesses a single genomic molecule.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29562/1/0000650.pd

Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries

BACKGROUND: The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae). Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. RESULTS: To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults) and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato mottle virus (ToMoV). In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production of eggs. CONCLUSION: This is the first functional genomics project involving a hemipteran (Homopteran) insect from the subtropics/tropics. The B. tabaci sequence database now provides an important tool to initiate identification of whitefly genes involved in development, behaviour, and B. tabaci-mediated begomovirus transmission

Discovering Host Genes Involved in the Infection by the Tomato Yellow Leaf Curl Virus Complex and in the Establishment of Resistance to the Virus Using Tobacco Rattle Virus-based Post Transcriptional Gene Silencing

The development of high-throughput technologies allows for evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infection of susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, which over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty-nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the 25 genes studied so far, the silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicates that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular mechanisms underlying plant infection and resistance to infection by begomoviruses