Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis.

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

Inoculation of Tomato With Plant Growth Promoting Rhizobacteria Affects the Tomato-Potato Psyllid-Candidatus Liberibacter Solanacearum Interactions.

The Rio Grande Valley (RGV) in southern Texas is well-suited for vegetable production due to its relatively mild/warm weather conditions in the fall and winter. Consequently, insects inflict year-round, persistent damage to crops in the RGV and regions with similar climate. Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), commonly known as the potato psyllid, is a known vector of Candidatus Liberibacter solanacearum (CLso) (Hyphomicrobiales: Rhizobiaceae), a fastidious phloem-limited bacterium associated to vein-greening in tomatoes and Zebra Chip in potatoes. Vector control is the primary approach of integrated pest management (IPM) strategies that aim to prevent plant diseases in commercial agricultural systems. However, resistance-selective pressures that decrease the effectiveness of chemical control (insecticide) applications over time are of increasing concern. Therefore, we explore an ecological approach to devising alternative IPM methodologies to manage the psyllid-transmitted CLso pathogen to supplement existing chemical products and application schedules without increasing resistance. In this study, our objective was to examine the effects of plant-growth promoting rhizobacteria (PGPR) on host-vector-pathogen interactions. Soil-drench applications of PGPRs to Solanum lycopersicum (Solanales: Solanaceae) seedlings revealed structural and possible physiological changes to the plant host and indirect changes on psyllid behavior: host plants had increased length and biomass of roots and exhibited delayed colonization by CLso, while psyllids displayed changes in parental (F0) psyllid behavior (orientation and oviposition) in response to treated hosts and in the sex ratio of their progeny (F1). Based on our results, we suggest that PGPR may have practical use in commercial tomato production.

Cry1Ba1-mediated toxicity of transgenic Bergera koenigii and Citrus sinensis to the Asian citrus psyllid Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan and Valencia expressing Cry1Ba1 exhibited a "delayed senescence" phenotype, similar to observations made for transgenic B. koenigii. In bioassays, significant reductions in the survival of adult psyllids were noted on transgenic B. koenigii and Valencia sweet orange plants expressing Cry1Ba1, but not on transgenic Duncan grapefruit or Carrizo citrange. In contrast to psyllids fed on wild type plants, the gut epithelium of psyllids fed on transgenic plants was damaged, consistent with the mode of action of Cry1Ba1. These results indicate that the transgenic expression of a bacterial pesticidal protein in B. koenigii and Valencia sweet orange offers a viable option for management of D. citri, that may contribute to solutions that counter citrus greening disease.

CLIBASIA_00460 Disrupts Hypersensitive Response and Interacts with Citrus Rad23 Proteins.

'Candidatus Liberibacter asiaticus' (CLas) is a bacterium that causes Huanglongbing, also known as citrus greening, in citrus plants. 'Candidatus Liberibacter solanacearum' (Lso) is a close relative of CLas and in the US it infects solanaceous crops, causing zebra chip disease in potato. Previously, we have identified the Lso hypothetical protein effector 1 (Lso-HPE1). This protein uses a signal peptide for secretion; disrupts programmed cell death; and interacts with tomato RAD23c, d, and e proteins, but not with RAD23a. In this study, we evaluated whether CLIBASIA_00460, the CLas homolog of Lso-HPE1 interacted with citrus RAD23 proteins and disrupted their programmed cell death. Based on the yeast two-hybrid assay results, CLIBASIA_00460 interacted with citrus RAD23c and RAD23d, but not with citrus RAD23b. These results were confirmed using bimolecular fluorescence complementation assays, which showed that these interactions occurred in cell puncta, but not in the nucleus or cytoplasm. Additionally, CLIBASIA_00460 was able to disrupt the PrfD1416V-induced hypersensitive response. Therefore, based on the similar interactions between Lso-HPE1 and CLIBASIA_00460 with the host RAD23 proteins and their ability to inhibit cell death in plants, we propose that these effectors may have similar functions during plant infection.

Identification and characterization of genes differentially expressed in cherimoya (Annona cherimola Mill) after exposure to chilling injury conditions.

Cherimoyas (Annona cherimola), like other subtropical/tropical fruits, are susceptible to damage from exposure to temperatures between 0 and 5 °C (chilling injury, CI), which may affect fruit quality. To increase our understanding of the molecular mechanisms involved in the CI response, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. In this work, we obtained 75 genes that could potentially be involved in the CI response. The CI induced activation of genes that are involved in a range of metabolic pathways, such as primary metabolism, transport, and endomembrane traffic, among others. We also characterized the expression of 12 selected genes in different A. cherimola tissues by polymerase chain reaction (PCR), and we confirmed the differential expression of a subset in CI fruits by real-time quantitative PCR (qPCR). The expression of six A. cherimola genes: annexin (AcAnex), UDP-glucose pyrophosphorylase (AcUGP), syntaxin of plants 71 (AcSyp71), 1-aminocyclopropane-1-carboxylic-acid synthase (AcACS), ubiquitin carrier-like protein (AcUCP), and enolase (AcEnol), was up-regulated after cold storage for 12 days at 0 °C. These results imply that selected genes could be related to the development of internal browning observed in cherimoyas after exposure to CI conditions. The information generated in this study provides new clues that may aid in understanding the cherimoya ripening process.