Structural and functional insights into the ATP-binding cassette transporter family in the corn planthopper, Peregrinus maidis.

The corn planthopper, Peregrinus maidis, is an economically important pest of maize and sorghum. Its feeding behaviour and the viruses it transmits can significantly reduce crop yield. The control of P. maidis and its associated viruses relies heavily on insecticides. However, control has proven difficult due to limited direct exposure of P. maidis to insecticides and rapid development of resistance. As such, alternative control methods are needed. In the absence of a genome assembly for this species, we first developed transcriptomic resources. Then, with the goal of finding targets for RNAi-based control, we identified members of the ATP-binding cassette transporter family and targeted specific members via RNAi. PmABCB_160306_3, PmABCE_118332_5 and PmABCF_24241_1, whose orthologs in other insects have proven important in development, were selected for knockdown. We found that RNAi-mediated silencing of PmABCB_160306_3 impeded ovary development; disruption of PmABCE_118332_5 resulted in localized melanization; and knockdown of PmABCE_118332_5 or PmABCF_24241_1 each led to high mortality within five days. Each phenotype is similar to that found when targeting the orthologous gene in other species and it demonstrates their potential for use in RNAi-based P. maidis control. The transcriptomic data and RNAi results presented here will no doubt assist with the development of new control methods for this pest.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.

The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Diversity and epidemiology of plant rhabdoviruses.

Plant rhabdoviruses are recognized by their large bacilliform particles and for being able to replicate in both their plant hosts and arthropod vectors. This review highlights selected, better studied examples of plant rhabdoviruses, their genetic diversity, epidemiology and interactions with plant hosts and arthropod vectors: Alfalfa dwarf virus is classified as a cytorhabdovirus, but its multifunctional phosphoprotein is localized to the plant cell nucleus. Lettuce necrotic yellows virus subtypes may differentially interact with their aphid vectors leading to changes in virus population diversity. Interactions of rhabdoviruses that infect rice, maize and other grains are tightly associated with their specific leafhopper and planthopper vectors. Future outbreaks of vector-borne nucleorhabdoviruses may be predicted based on a world distribution map of the insect vectors. The epidemiology of coffee ringspot virus and its Brevipalpus mite vector is illustrated highlighting the symptomatology and biology of a dichorhavirus and potential impacts of climate change on its epidemiology.

Plant rhabdoviruses-their origins and vector interactions.

Classical plant rhabdoviruses infect monocot and dicot plants, have unsegmented negative-sense RNA genomes and have been taxonomically classified in the genera Cytorhabdovirus and Nucleorhabdovirus. These viruses replicate in their hemipteran vectors and are transmitted in a circulative-propagative mode and virus infection persists for the life of the insect. Based on the discovery of numerous novel rhabdoviruses in arthropods during metagenomic studies and extensive phylogenetic analyses of the family Rhabdoviridae, it is hypothesized that plant-infecting rhabdoviruses are derived from insect viruses. Analyses of viral gene function in plants and insects is beginning to reveal conserved and unique biology for these plant viruses in the two diverse hosts. New tools for insect molecular biology and infectious clones for plant rhabdoviruses are increasing our understanding of the lifestyles of these viruses.

Global gene regulation in tomato plant (Solanum lycopersicum) responding to vector (Bactericera cockerelli) feeding and pathogen ('Candidatus Liberibacter solanacearum') infection.

KEY MESSAGE: Different responses are elicited in tomato plants by Bactericera cockerelli harboring or not the pathogen 'Candidatus Liberibacter solanacearum'. 'Candidatus Liberibacter solanacearum' (Lso) has emerged as a major pathogen of crops worldwide. This bacterial pathogen is transmitted by Bactericera cockerelli, the tomato psyllid, to solanaceous crops. In this study, the transcriptome profiles of tomato (Solanum lycopersicum) exposed to B. cockerelli infestation and Lso infection were evaluated at 1, 2 and 4 weeks following colonization and/or infection. The plant transcriptional responses to Lso-negative B. cockerelli were different than plant responses to Lso-positive B. cockerelli. The comparative transcriptome analyses of plant responses to Lso-negative B. cockerelli revealed the up-regulation of genes associated with plant defenses regardless of the time-point. In contrast, the general responses to Lso-positive B. cockerelli and Lso-infection were temporally different. Infected plants down-regulated defense genes at week one while delayed the up-regulation of the defense genes until weeks two and four, time points in which early signs of disease development were also detected in the transcriptional response. For example, infected plants regulated carbohydrate metabolism genes which could be linked to the disruption of sugar distribution usually associated with Lso infection. Also, infected plants down-regulated photosynthesis-related genes potentially resulting in plant chlorosis, another symptom associated with Lso infection. Overall, this study highlights that tomato plants induce different sets of genes in response to different stages of B. cockerelli infestation and Lso infection. This is the first transcriptome study of tomato responses to B. cockerelli and Lso, a first step in the direction of finding plant defense genes to enhance plant resistance.

The influence of bacteria on multitrophic interactions among plants, psyllids, and pathogen.

The recent emergence of several plant diseases caused by psyllid-borne bacterial pathogens worldwide (Candidatus Liberibacter spp.) has created renewed interest on the interaction between psyllids and bacteria. In spite of these efforts to understand psyllid association with bacteria, many aspects of their interactions remain poorly understood. As more organisms are studied, subtleties on the molecular interactions as well as on the effects of the bacteria on the psyllid host are being uncovered. Additionally, psyllid-borne bacterial phytopathogens can also affect the host plant, which in turn can impact psyllid physiology and behavior. Here, we review the current literature on different aspects of the influence of bacteria on multitrophic interactions among plants, psyllids, and pathogens. We then highlight gaps that need to be addressed to advance this field, which can have significant implications for controlling these newly emergent and other plant diseases.

Interactions "Candidatus Liberibacter solanacearum"-Bactericera cockerelli: Haplotype Effect on Vector Fitness and Gene Expression Analyses.

"Candidatus Liberibacter solanacearum" (Lso) has emerged as a serious threat world-wide. Five Lso haplotypes have been identified so far. Haplotypes A and B are present in the Americas and/or New Zealand, where they are vectored to solanaceous plants by the potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). The fastidious nature of these pathogens has hindered the study of the interactions with their eukaryotic hosts (vector and plant). To understand the strategies used by these pathogens to infect their vector, the effects of each Lso haplotype (A or B) on psyllid fitness was investigated, and genome-wide transcriptomic and RT-qPCR analyses were performed to evaluate Lso gene expression in association with its vector. Results showed that psyllids infected with haplotype B had significantly lower percentage of nymphal survival compared to psyllids infected with haplotype A. Although overall gene expression across Lso genome was similar between the two Lso haplotypes, differences in the expression of key candidate genes were found. Among the 16 putative type IV effector genes tested, four of them were differentially expressed between Lso haplotypes, while no differences in gene expression were measured by qPCR or transcriptomic analysis for the rest of the genes. This study provides new information regarding the pathogenesis of Lso haplotypes in their insect vector.

The evolutionary strategies of plant defenses have a dynamic impact on the adaptations and interactions of vectors and pathogens.

Plants have evolved and diversified to reduce the damages imposed by infectious pathogens and herbivorous insects. Living in a sedentary lifestyle, plants are constantly adapting to their environment. They employ various strategies to increase performance and fitness. Thus, plants developed cost-effective strategies to defend against specific insects and pathogens. Plant defense, however, imposes selective pressure on insects and pathogens. This selective pressure provides incentives for pathogens and insects to diversify and develop strategies to counter plant defense. This results in an evolutionary arms race among plants, pathogens and insects. The ever-changing adaptations and physiological alterations among these organisms make studying plant-vector-pathogen interactions a challenging and fascinating field. Studying plant defense and plant protection requires knowledge of the relationship among organisms and the adaptive strategies each organism utilize. Therefore, this review focuses on the integral parts of plant-vector-pathogen interactions in order to understand the factors that affect plant defense and disease development. The review addresses plant-vector-pathogen co-evolution, plant defense strategies, specificity of plant defenses and plant-vector-pathogen interactions. Improving the comprehension of these factors will provide a multi-dimensional perspective for the future research in pest and disease management.