Tomato bushy stunt virus (TBSV) is a positive-sense single-stranded RNA virus. It encodes five open reading frames (ORFs), including two nested genes, expressing movement-associated proteins. One of these proteins, P22, interacts with a host transcription factor containing a homeodomain leucine-zipper motif, known as HFi22. Similar proteins of this type traffic their RNA from cell-to-cell, suggesting the possiblity that HFi22 is involved in the cell-to-cell movement of TBSV RNA. To further characterize the nature of the interaction between P22 and HFi22 on the cellular level, cellular fractionation experiments were conducted. To investigate the functional role of HFi22 in viral movement I attempted to inactivate its expression using a virus induced gene silencing system with a Tobacco rattle virus (TRV) vector. A final objective was based on the notion that different hosts can impact the stability of viruses used to express foreign genes of biotechnological interest. To compare virus stability in different hosts, TBSV expressing the green fluorescent protein (GFP) was inoculated onto various TBSV hosts, and infected leaf tissue was then used as inoculum to be rubbed onto a local lesion host. This technique made it possible to quantify the number of fluorescent foci versus total lesions. Results obtained for the first objective indicate that P22 and HFi22 co-fractionate in nucleus and membrane-enriched samples. In addition, it was found that HFi22 is largely conserved through a wide variety of plant species but not in lettuce, which was found to be compromised for effective virus spread. Control experiments for the second objective showed that plants were successfully silenced with TRV carrying the phytoene desaturase (PDS) gene resulting in photobleaching, however attempts to silence HFi22 have not yielded conclusive results. The results obtained for the third objective indicate there is a difference in how efficiently a foreign gene insert is maintained by TBSV in different host plants. In summary, the overall results of this research showed that host factors influence the host-virus interaction but their exact contributions remain elusive.

Tomato bushy stunt virus (TBSV) is a positive-sense single-stranded RNA virus. It encodes five open reading frames (ORFs), including two nested genes, expressing movement-associated proteins. One of these proteins, P22, interacts with a host transcription factor containing a homeodomain leucine-zipper motif, known as HFi22. Similar proteins of this type traffic their RNA from cell-to-cell, suggesting the possiblity that HFi22 is involved in the cell-to-cell movement of TBSV RNA. To further characterize the nature of the interaction between P22 and HFi22 on the cellular level, cellular fractionation experiments were conducted. To investigate the functional role of HFi22 in viral movement I attempted to inactivate its expression using a virus induced gene silencing system with a Tobacco rattle virus (TRV) vector. A final objective was based on the notion that different hosts can impact the stability of viruses used to express foreign genes of biotechnological interest. To compare virus stability in different hosts, TBSV expressing the green fluorescent protein (GFP) was inoculated onto various TBSV hosts, and infected leaf tissue was then used as inoculum to be rubbed onto a local lesion host. This technique made it possible to quantify the number of fluorescent foci versus total lesions. Results obtained for the first objective indicate that P22 and HFi22 co-fractionate in nucleus and membrane-enriched samples. In addition, it was found that HFi22 is largely conserved through a wide variety of plant species but not in lettuce, which was found to be compromised for effective virus spread. Control experiments for the second objective showed that plants were successfully silenced with TRV carrying the phytoene desaturase (PDS) gene resulting in photobleaching, however attempts to silence HFi22 have not yielded conclusive results. The results obtained for the third objective indicate there is a difference in how efficiently a foreign gene insert is maintained by TBSV in different host plants. In summary, the overall results of this research showed that host factors influence the host-virus interaction but their exact contributions remain elusive.

Two Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation. A primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles. VIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized. To characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC. Characterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.

Tombusviruses are single, positive strand RNA viruses of plants, often associated with parasitic defective interfering (DI) RNAs. Two viral- coded gene products, namely p33 and p92, are required for tombusvirus replication. The overlapping domains of p33 and p92 contain an arginine/proline-rich (RPR) RNA binding motif. In this study, the role of RPR motif and viral RNA in tombusvirus replication and recombination, as well as involvement of viral RNA in tombusvirus replicase assembly was examined. Using site-directed mutagenesis I generated a series of RPR mutants of Cucumber necrosis tombusvirus (CNV). Analysis of RPR mutants defined that wild type RPR motif, especially two of the four arginines, were required for efficient RNA binding in vitro, for replication of tombusviruses, their associated DI RNAs, subgenomic (sg)RNA synthesis and DI RNA recombination in vivo. Experiments using a two-component tombusvirus replication system showed that RPR motif is critical for functions of both p33 and p92 in replication, but its role in these proteins might not be identical. Recombination studies using a novel tombusvirus three-component system revealed that mutations in RPR motif of p33 replicase protein resulted in an altered viral RNA recombination rate. Identified DI RNA recombinants were mostly imprecise, with recombination sites clustered around a replication enchancer and an additional putative cis-acting element that might facilitate the template switching events by the tombusvirus replicase. To study the role of RNA during the assembly of functional tombusvirus replicase, recombinant CNV replicase that showed similar properties to plant-derived CNV replicase was purified from Saccharomyces cerevisiae. When in addition to p33 and p92 proteins DI RNA was co-expressed in yeast cells, the isolated replicase activity was increased ~40 fold. Further studies defined RNA motifs within two short DI RNA regions that enhanced active CNV replicase formation. In summary, this study showed that the conserved RNA binding motif of the tombusvirus replicase proteins and viral RNA are involved in replicase assembly, viral RNA replication, subgenomic RNA synthesis and RNA recombination. This data shed new light on the complex roles of the viral elements in replication, and will help future studies aimed at interfering with viral infections.

Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare due to the devastating diseases these viruses inflict on humans, animals and plants. Rapid viral RNA evolution is frequently due to RNA recombination, which can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. We have demonstrated that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. Our results support the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses. To test if host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes ~80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from TBSV. The genome-wide screen led to the identification of five host genes, whose absence resulted in rapid generation of novel viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence hosts become viral recombination hotbeds. Four of the five recombination suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. Overall, our results demonstrate for the first time that a set of host genes have major effect on RNA virus recombination and evolution. Replication of the non-segmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. We have demonstrated that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Our findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions. / Fitoplasmas são bactérias transmitidas de forma persistente propagativa por insetos vetores, havendo interações diretas e indiretas envolvendo tais organismos. Para entender algumas dessas interações no caso do enfezamento vermelho do milho, associado ao \'maize bushy stunt phytoplasma\' (MBPS) e à cigarrinha vetora Dalbulus maidis (Hemiptera: Cicadellidae), foram desenvolvidas duas linhas de trabalho. Na primeira determinou-se o efeito indireto de MBSP no comportamento e biologia do vetor, enquanto que na segunda estudaram-se as interações diretas do fitoplasma com o vetor durante sua movimentação através de órgãos internos e associação com a microbiota do inseto. O efeito indireto no comportamento foi demostrado em testes de escolha nos quais se variou o período de incubação de MBSP na planta (plantas com sintomas iniciais ou avançados da doença, e plantas infectadas assintomáticas) sobre a preferência para pouso e oviposição pelo vetor. O efeito indireto na biologia do D. maidis foi estabelecido em dois experimentos de tabela de vida, alimentando o inseto durante seu ciclo de vida em plantas de milho sadias ou plantas infectadas com o fitoplasma em dois tempos de incubação (plantas com sintomas avançados da doença e plantas infectadas assintomáticas). Os testes de escolha mostraram que a preferência de D. maidis para pouso e oviposição em plantas infectadas por MBSP em relação a plantas sadias depende do período de incubação do patógeno na planta. A cigarrinha preferiu plantas infectadas por MBSP na fase assintomática da doença, mas rejeitou plantas com sintomas avançados. Ocorreu aquisição do fitoplasma pelo vetor em plantas assintomáticas a partir de 3 dias após a inoculação, mas a eficiência de transmissão aumentou após 14 dias de incubação do patógeno na planta-fonte, e diminuiu o tempo para expressão de sintomas nas plantas-teste inoculadas. Os resultados sugerem que MBSP modula a preferência do vetor para plantas infectadas no estágio inicial da cultura de modo a permitir sua rápida disseminação. O efeito da infecção de plantas por MBSP na biologia de D. maidis mostrou ser neutro para a maioria dos parâmetros biológicos estimados; houve menor taxa líquida de reprodução (Ro), que foi compensada por maior razão sexual, para insetos criados em plantas com sintomas avançados. Verificou-se, por PCR, aquisição do fitoplasma por todos os estádios ninfais do vetor e sua presença em órgãos reprodutivos dos adultos. Por microscopia eletrônica de transmissão, observaram-se células do tipo fitoplasma no lúmen, microvilosidades e células epiteliais do mesêntero de D. maidis, sugerindo que o MBSP penetra no epitélio através das microvilosidades. No epitélio intestinal, foram observadas massas de células do tipo fitoplasma próximas a mitocôndrias e células bacterianas, possíveis endossimbiontes. Na hemocele, também foram observadas células do tipo fitoplasma agrupadas numa matriz, em associação com bactérias similares às observadas no intestino. Associações semelhantes foram observadas na glândula salivar. A técnica de FISH revelou uma variação na riqueza e abundância das espécies na da microbiota no mesêntero e glândula salivar de D. maidis em função do tempo após a aquisição de MBSP. A abundância de Sulcia sp., Cardinium sp. e eubacteria aumentou, enquanto que a de Rickettsia sp. decresceu. A associação frequente dessas bactérias com fitoplasma em alguns tecidos de D. maidis sugere que endossimbiontes possam ter um papel nas interações fitoplasma-vetor.

O milho (Zea mays L.) é uma cultura de grande importância para o agronegócio brasileiro, que enfrenta diversos problemas fitossanitários. Dentre as doenças destaca-se o enfezamento vermelho, que está associado ao molicute fitopatogênico, \'maize bushy stunt phytoplasma\' (MBSP). Este fitoplasma é transmitido por um grupo restrito de cicadelídeos (Hemiptera: Cicadellidae), constituído principalmente por espécies do gênero Dalbulus; no Brasil é relatada a presença de apenas uma espécie vetora, a cigarrinha-do-milho, Dalbulus maidis (DeLong & Wolcott). A cigarrinha-do-milho possui profunda associação e coevolução com o milho, sendo considerada específica dessa cultura. No México há relatos de D. maidis se desenvolvendo em espécies selvagens do gênero Zea e Tripsacum. Mas no Brasil a única planta hospedeira conhecida de D. maidis é o milho. Portanto, não se sabe como esta cigarrinha, assim como o fitoplasma por ela transmitido, sobrevivem ao longo período de entressafra que ocorre em algumas regiões brasileiras. Estudos têm demonstrado que a presença de fitopatógenos na planta e nos insetos e as alterações por eles causadas na fisiologia dos mesmos, podem melhorar desempenho biológico do inseto vetor. No México, MBSP é altamente patogênico a outras espécies de cigarrinhas do gênero Dalbulus que se alimentam no milho e competem com D. maidis. Considerando-se que no Brasil outras espécies de cigarrinhas apresentam pequenas populações em milho, postulase que este fitoplasma as afete negativamente, reduzindo sua capacidade de competição com D. maidis. O presente trabalho investigou possíveis plantas hospedeiras alternativas do inseto e do fitoplasma, outros possíveis vetores do MBSP, e o efeito do fitoplasma na sobrevivência de D. maidis e em alguns parâmetros biológicos desse vetor e do cicadelídeo Agallia albidula Uhler, espécie não vetora, presente na vegetação espontânea da cultura do milho. Dez espécies de plantas daninhas em milharais foram submetidas à inoculação de MBSP com adultos infectivos de D. maidis, mas em nenhuma delas foi detectada a infecção pelo fitoplasma, indicando que o fitoplasma possui alta especificidade em relação a plantas hospedeiras. Duas espécies de cicadelídeos comuns na vegetação rasteira de cultivos de milho, A. albidula e Planicephalus flavicosta (Stål) foram testadas como vetores de MBSP, e nenhuma delas foi capaz de transmitir o patógeno para o milho, indicando que a especificidade de vetores também é elevada. A infecção de D. maidis por MBSP afetou negativamente a sobrevivência de adultos deste inseto vetor. Por outro lado, plantas de milho infectadas por MBSP estimularam a oviposição das cigarrinhas D. maidis e A. albidula. Entretanto, a sobrevivência de ninfas de A. albidula foi muito baixa tanto em plantas sadias quanto em plantas infectadas pelo fitoplasma, indicando que o milho, independentemente do fitoplasma, não é um bom hospedeiro para desenvolvimento de A. albidula. / Corn (Zea mays L.) is an important crop for Brazilian agribusiness, but it is affected by various pests and diseases. A stunting disease associated with the phytopathogenic mollicute, maize bushy stunt phytoplasma (MBPS), is a one of the main problems. This phytoplasma is transmitted by a narrow range of leafhoppers (Hemiptera: Cicadellidae), mainly in the genus Dalbulus. In Brazil, the only known vector is the corn leafhopper, Dalbulus maidis (DeLong & Walcott). The corn leafhopper has a deep association and co-evolution with corn, and it is thought to be specific to this crop. In Mexico there are reports of D. maidis developing on wild species of Zea and Tripsacum. Because corn is the only known host of D. maidis in Brazil, it is unknown how this leafhopper and MBSP survive the long corn off-season that occurs in some Brazilian growing regions. Studies have shown that the presence of pathogens in plants and insects and the physiological changes caused by them can improve the biological performance of insect vectors. In Mexico, MBSP is highly pathogenic to some Dalbulus species that feed on corn and compete with D. maidis. Considering that in Brazil other leafhopper species have small populations in corn, it is postulated that MBSP may negatively affect them and reduce their ability to compete with D. maidis. The current study investigated possible alternative host plants of MBSP and D. maidis, other possible leafhopper vectors of MBSP and the effects of the phytoplasma on D. maidis survival and in some biological parameters of this vector and of Agallia albidula Uhler, a non-vector leafhopper present in the weedy vegetation of corn fields. Ten weed species commonly present in corn fields were inoculated with MBSP by infective adults of D. maidis, but none of them became infected with the phytoplasma, suggesting that MBSP has high host plant specificity. Two leafhopper species that inhabit the weedy vegetation of corn fields, A. albidula e Planicephalus flavicosta (Stål), were tested as vectors of MBSP, but none of them was able to transmit the pathogen to corn, indicating that the vector specificity of this phytoplasma is also high. Infection of D. maidis by MBSP negatively affected adult survival. On the other hand, infection of corn plants by MBSP induced higher oviposition by D. maidis and A. albidula. However, the survival of A. albidula nymphs was very low not only on infected plants, but also on healthy corn plants, indicating corn is not a suitable developmental host for A. albidula, regardless of the phytoplasma infection.

O milho (Zea mays L.) é uma cultura de grande importância para o agronegócio brasileiro, que enfrenta diversos problemas fitossanitários. Dentre as doenças destaca-se o enfezamento vermelho, que está associado ao molicute fitopatogênico, \'maize bushy stunt phytoplasma\' (MBSP). Este fitoplasma é transmitido por um grupo restrito de cicadelídeos (Hemiptera: Cicadellidae), constituído principalmente por espécies do gênero Dalbulus; no Brasil é relatada a presença de apenas uma espécie vetora, a cigarrinha-do-milho, Dalbulus maidis (DeLong & Wolcott). A cigarrinha-do-milho possui profunda associação e coevolução com o milho, sendo considerada específica dessa cultura. No México há relatos de D. maidis se desenvolvendo em espécies selvagens do gênero Zea e Tripsacum. Mas no Brasil a única planta hospedeira conhecida de D. maidis é o milho. Portanto, não se sabe como esta cigarrinha, assim como o fitoplasma por ela transmitido, sobrevivem ao longo período de entressafra que ocorre em algumas regiões brasileiras. Estudos têm demonstrado que a presença de fitopatógenos na planta e nos insetos e as alterações por eles causadas na fisiologia dos mesmos, podem melhorar desempenho biológico do inseto vetor. No México, MBSP é altamente patogênico a outras espécies de cigarrinhas do gênero Dalbulus que se alimentam no milho e competem com D. maidis. Considerando-se que no Brasil outras espécies de cigarrinhas apresentam pequenas populações em milho, postulase que este fitoplasma as afete negativamente, reduzindo sua capacidade de competição com D. maidis. O presente trabalho investigou possíveis plantas hospedeiras alternativas do inseto e do fitoplasma, outros possíveis vetores do MBSP, e o efeito do fitoplasma na sobrevivência de D. maidis e em alguns parâmetros biológicos desse vetor e do cicadelídeo Agallia albidula Uhler, espécie não vetora, presente na vegetação espontânea da cultura do milho. Dez espécies de plantas daninhas em milharais foram submetidas à inoculação de MBSP com adultos infectivos de D. maidis, mas em nenhuma delas foi detectada a infecção pelo fitoplasma, indicando que o fitoplasma possui alta especificidade em relação a plantas hospedeiras. Duas espécies de cicadelídeos comuns na vegetação rasteira de cultivos de milho, A. albidula e Planicephalus flavicosta (Stål) foram testadas como vetores de MBSP, e nenhuma delas foi capaz de transmitir o patógeno para o milho, indicando que a especificidade de vetores também é elevada. A infecção de D. maidis por MBSP afetou negativamente a sobrevivência de adultos deste inseto vetor. Por outro lado, plantas de milho infectadas por MBSP estimularam a oviposição das cigarrinhas D. maidis e A. albidula. Entretanto, a sobrevivência de ninfas de A. albidula foi muito baixa tanto em plantas sadias quanto em plantas infectadas pelo fitoplasma, indicando que o milho, independentemente do fitoplasma, não é um bom hospedeiro para desenvolvimento de A. albidula. / Corn (Zea mays L.) is an important crop for Brazilian agribusiness, but it is affected by various pests and diseases. A stunting disease associated with the phytopathogenic mollicute, maize bushy stunt phytoplasma (MBPS), is a one of the main problems. This phytoplasma is transmitted by a narrow range of leafhoppers (Hemiptera: Cicadellidae), mainly in the genus Dalbulus. In Brazil, the only known vector is the corn leafhopper, Dalbulus maidis (DeLong & Walcott). The corn leafhopper has a deep association and co-evolution with corn, and it is thought to be specific to this crop. In Mexico there are reports of D. maidis developing on wild species of Zea and Tripsacum. Because corn is the only known host of D. maidis in Brazil, it is unknown how this leafhopper and MBSP survive the long corn off-season that occurs in some Brazilian growing regions. Studies have shown that the presence of pathogens in plants and insects and the physiological changes caused by them can improve the biological performance of insect vectors. In Mexico, MBSP is highly pathogenic to some Dalbulus species that feed on corn and compete with D. maidis. Considering that in Brazil other leafhopper species have small populations in corn, it is postulated that MBSP may negatively affect them and reduce their ability to compete with D. maidis. The current study investigated possible alternative host plants of MBSP and D. maidis, other possible leafhopper vectors of MBSP and the effects of the phytoplasma on D. maidis survival and in some biological parameters of this vector and of Agallia albidula Uhler, a non-vector leafhopper present in the weedy vegetation of corn fields. Ten weed species commonly present in corn fields were inoculated with MBSP by infective adults of D. maidis, but none of them became infected with the phytoplasma, suggesting that MBSP has high host plant specificity. Two leafhopper species that inhabit the weedy vegetation of corn fields, A. albidula e Planicephalus flavicosta (Stål), were tested as vectors of MBSP, but none of them was able to transmit the pathogen to corn, indicating that the vector specificity of this phytoplasma is also high. Infection of D. maidis by MBSP negatively affected adult survival. On the other hand, infection of corn plants by MBSP induced higher oviposition by D. maidis and A. albidula. However, the survival of A. albidula nymphs was very low not only on infected plants, but also on healthy corn plants, indicating corn is not a suitable developmental host for A. albidula, regardless of the phytoplasma infection.

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions. / Fitoplasmas são bactérias transmitidas de forma persistente propagativa por insetos vetores, havendo interações diretas e indiretas envolvendo tais organismos. Para entender algumas dessas interações no caso do enfezamento vermelho do milho, associado ao \'maize bushy stunt phytoplasma\' (MBPS) e à cigarrinha vetora Dalbulus maidis (Hemiptera: Cicadellidae), foram desenvolvidas duas linhas de trabalho. Na primeira determinou-se o efeito indireto de MBSP no comportamento e biologia do vetor, enquanto que na segunda estudaram-se as interações diretas do fitoplasma com o vetor durante sua movimentação através de órgãos internos e associação com a microbiota do inseto. O efeito indireto no comportamento foi demostrado em testes de escolha nos quais se variou o período de incubação de MBSP na planta (plantas com sintomas iniciais ou avançados da doença, e plantas infectadas assintomáticas) sobre a preferência para pouso e oviposição pelo vetor. O efeito indireto na biologia do D. maidis foi estabelecido em dois experimentos de tabela de vida, alimentando o inseto durante seu ciclo de vida em plantas de milho sadias ou plantas infectadas com o fitoplasma em dois tempos de incubação (plantas com sintomas avançados da doença e plantas infectadas assintomáticas). Os testes de escolha mostraram que a preferência de D. maidis para pouso e oviposição em plantas infectadas por MBSP em relação a plantas sadias depende do período de incubação do patógeno na planta. A cigarrinha preferiu plantas infectadas por MBSP na fase assintomática da doença, mas rejeitou plantas com sintomas avançados. Ocorreu aquisição do fitoplasma pelo vetor em plantas assintomáticas a partir de 3 dias após a inoculação, mas a eficiência de transmissão aumentou após 14 dias de incubação do patógeno na planta-fonte, e diminuiu o tempo para expressão de sintomas nas plantas-teste inoculadas. Os resultados sugerem que MBSP modula a preferência do vetor para plantas infectadas no estágio inicial da cultura de modo a permitir sua rápida disseminação. O efeito da infecção de plantas por MBSP na biologia de D. maidis mostrou ser neutro para a maioria dos parâmetros biológicos estimados; houve menor taxa líquida de reprodução (Ro), que foi compensada por maior razão sexual, para insetos criados em plantas com sintomas avançados. Verificou-se, por PCR, aquisição do fitoplasma por todos os estádios ninfais do vetor e sua presença em órgãos reprodutivos dos adultos. Por microscopia eletrônica de transmissão, observaram-se células do tipo fitoplasma no lúmen, microvilosidades e células epiteliais do mesêntero de D. maidis, sugerindo que o MBSP penetra no epitélio através das microvilosidades. No epitélio intestinal, foram observadas massas de células do tipo fitoplasma próximas a mitocôndrias e células bacterianas, possíveis endossimbiontes. Na hemocele, também foram observadas células do tipo fitoplasma agrupadas numa matriz, em associação com bactérias similares às observadas no intestino. Associações semelhantes foram observadas na glândula salivar. A técnica de FISH revelou uma variação na riqueza e abundância das espécies na da microbiota no mesêntero e glândula salivar de D. maidis em função do tempo após a aquisição de MBSP. A abundância de Sulcia sp., Cardinium sp. e eubacteria aumentou, enquanto que a de Rickettsia sp. decresceu. A associação frequente dessas bactérias com fitoplasma em alguns tecidos de D. maidis sugere que endossimbiontes possam ter um papel nas interações fitoplasma-vetor.