Hemipteran insects are a significant threat to food security in California and worldwide; their piercing-sucking mouthparts make them difficult for host plants to perceive. Of these insect pests, the whitefly (Bemisia tabaci) is a cosmopolitan pest which stunts plant growth and development, vectors viruses, and also secretes honeydew which can result in sooty mold growth on host plants. Whiteflies are extant on all continents except Antarctica and climate change increases the propensity superabundant whitefly populations will be more common worldwide over time. Whiteflies are difficult to control as the release of natural enemies in crops has limited effectiveness and whiteflies circumvent the pesticides by rapidly adapting by developing insecticide resistance. For this reason, integrated pest management (IPM) programs centered around host plant resistance (HPR) may be the most effective means of controlling whiteflies. An effective, nymph-based whitefly-resistance mechanism was identified in alfalfa. While this mechanism may greatly inhibit whitefly population over generations, the polyploid nature and limited genomic resources in alfalfa make elucidating this resistance a challenge. Here, we describe whitefly-resistance found among three alfalfa populations. Upon screening 84 individual lines from the three populations (two resistant and one susceptible), we concluded whitefly resistance was multigenic as a continuous spectrum of phenotypes from highly resistance to highly susceptible in each population. We identified several highly resistant (R1, R2 and R3) and susceptible lines of alfalfa (S1) for further studies. Through a series of experiments exploring B. tabaci MEAM1, MED1 and NW1 behaviors, we determined the whitefly-resistance displayed in R1, R2 and R3 lines were distinct and both antibiosis and antixenosis were detected for all three whitefly species. MEAM1 nymph mortality was displayed in all R lines, while the nymph mortality mechanisms did not impact MED whitefly. In addition, differences in host-choice, adult longevity and fecundity on R1, R2, R3 and S1 lines were whitefly-species specific. To gain insights into the mechanisms of resistance deployed in R1 plants vs S1 plants, B. tabaci MEAM1 whitefly-infestation time courses in R1 and S1 plants were performed. To understand the phytohormone underpinnings of alfalfa’s defense response to whitefly, S1 alfalfa’s response to salicylic acid (SA) and jasmonic acid (JA) treatments was also evaluated. De novo transcriptomic assembly of these libraries led us to postulate alfalfa’s whitefly resistance mechanism is independent of SA, JA and abscisic acid (ABA) signaling and is ET-dependent. In addition, the downregulation of several pattern-triggered immunity receptors, suggests defense signaling in R1 plants is distinct for S1 plants and unique in the resistance responses reported to date in Hemipteran literature. In addition, R1 alfalfa have substantial difference in the expression of cutin, wax and suberin biosynthesis transcripts implicating the role of cuticle/cell wall alterations in R1’s whitefly resistance. Analysis of phytohormone-response libraries led us to conclude alfalfa’s SA and JA responses are distinct from Arabidopsis as there was no evidence for reciprocal regulation of SA and JA responses and a substantial number of genes are responsive to both hormones. Finally, unlike the previously characterized basal immunity response Arabidopsis to whiteflies, where JA has an important role in deterring nymph development, and there is little correlation between alfalfa’s whitefly response and SA- and/or JA-regulated genes. Collectively, these data provide the first insights into the alfalfa’s mechanism of resistance to the global pest Bemisia tabaci.

Plant leucine aminopeptidase A (LAP-A) modulates late wound responses and insectdefense. LAP-A is an aminopeptidase and chaperone in the chloroplast stroma. Based on the ability of LAP-A to modulate transcripts in the wound-response pathway, we postulated that LAP-A sends a retrograde signal(s) to the nucleus to regulate nuclear gene expression in response to wounding and MeJA treatments. We use wild-type, LapA-silenced and LapA-overexpressing plants to explore the global impact of LAP-A deficiency or ectopic expression on the tomato metabolome. This dissertation has revealed that LAP-A has multiple impacts on tomato metabolites by altering the levels of amino acids and secondary metabolites involved in plant defense. Given the link to sulfur metabolism and LAP-A’s ability to hydrolyze glutathione’s catabolic product Cys- Gly, the role of LAP-A in GSH metabolism using targeted metabolomics and the three genotypes was assessed. These wound-time course experiments were designed to simultaneously measure H2O2 levels. LAP-A did not regulate the levels of glutathione (GSH), the redox status of GSH, nor the levels of Cys-Gly, Cys or g-Glu-Cys. In the absence of LAP-A, H2O2 levels are elevated indicating that LAP-A is important for the control of ROS. H2O2 is a known retrograde signal used in defense. To elucidate LAPA’s possible mechanism(s) of action, the proteins that bind to LAP-A, but not to the highly related LAP-N, were identified. The 86 LAP-A-interacting proteins that reside in the chloroplast suggests that LAP-A may exert its effects via the major chloroplast redox hub associated with 2-Cys-peroxiredoxin. LAP-A binds to NADPH thioredoxin reductase- C (NTRC1), which provides the reducing power to 2-Cys-peroxiredoxin for clearing H2O2 from the chloroplast; a model is presented. Additional LAP-A-interacting proteins suggest a role for LAP-A in modulating the activity of the stromal Clp protease. Finally, this dissertation reports the first tomato stromal proteome. The 1254 stromal proteins identified were manually annotated. This is the first evidence for the chloroplast location of 550 tomato proteins. This study is foundational for current initiatives to understand the LAP-A- and MeJA-dependent changes in the tomato stromal proteome, which should provide additional insights into the global impacts of LAP-A during plant stress responses.

African whitefly Bemisia tabaci is a yield-limiting pest of the subsistence crop cassava through feeding and vectoring viral diseases. To address this issue, the African Cassava Whitefly Project is identifying whitefly resistance in South American cassava for selective breeding into African cassava. The collaborative efforts of project members at the International Center for Tropical Agriculture, UCR and the Royal Holloway University of London have focused on the genetic, transcriptomic and metabolic basis of resistance to Latin American whitefly Aleurotrachelus socialis in whitefly-resistant Ecuadorian cassava genotype ECU72. Here, the whitefly- and defense-hormone-responsive transcriptomes of ECU72 and its susceptible counterpart COL2246 have been defined for analysis and integration with these data sets. To characterize the cassava-whitefly interaction, we identified a core transcriptome response of Pathogenesis-related (PR) gene families to whitefly infestation in four whitefly-susceptible genotypes. Defense-hormone responses of COL2246 revealed that whitefly-responsive PR genes were mainly co-regulated by salicylic acid (SA) and jasmonic acid (JA). Cell-wall-related PR gene families were dominant in the responses to whiteflies and biotic stressors. PR gene phylogenies and biotic-stress-responsive transcriptomes revealed a possible selection in cassava for expansion of certain PR genes responsive to whiteflies and microbes. More global characterization of the cassava-whitefly interaction compared whitefly-, SA-, JA-, ethylene-, and abscisic acid (ABA)-responsive transcriptomes in ECU72 and COL2246. SA responses were faster and more prolonged in ECU72, and SA-responsive genes showed reciprocity between genotypes, suggesting importance of crosstalk regulators. Hormone responses during infestation revealed evidence of ABA-mediated resistance in ECU72 and SA-mediated susceptibility in COL2246. Comparisons between cassava and Arabidopsis revealed divergent transcriptome responses to SA and JA. Enrichment analyses of whitefly- and hormone-regulated genes showed that early infestation prompted cell-wall-based defenses in ECU72, while late infestation invoked SA-signaling and immunity defenses in COL2246. To identify whitefly defense genes, eQTL analysis was performed utilizing F1 progeny generated from crosses of ECU72 with whitefly-susceptible COL2246 or 60444. Criteria applied to eQTLs identified immunity, defense-signaling and cell-wall-related processes as possibly important in defense against whiteflies. eQTL-identified genes involved in ABA responses, monolignol biosynthesis, and chitin perception/response are proposed as possible whitefly resistance factors for evaluation in transgenic cassava.

In the absence of strong resistance mechanisms to control the world-wide pest Bemisia tabaci B, new methods for control must be derived from understanding the plant innate immune response to whiteflies. Using four Arabidopsis defense-signaling mutants, transcriptome and hormone treatment studies, it was shown that B. tabaci B activates SA-regulated defenses, suppresses JA-regulated defenses, and the JA-defenses are effective in slowing whitefly nymphal development.

Here we used the Electrical Penetration Graph (EPG) technique to determine the tissue location of resistance traits that influence B. tabaci B adult and nymph feeding in wild-type plants and in four defense mutants (cim10, cev1, coi1, and npr1). EPG studies measured 90 variables reflecting adult whitefly feeding behaviors. However, few variables differed significantly among the five genotypes and between pooled data of fast-development genotypes (cim10 and coi1) and slow nymph development genotypes (npr1 and cev1). These data indicated that the suite of variables that are most strongly associated with host plant acceptance/suitability were not consistently associated with how each genotype affected nymphal development. For most of the feeding variables where the ANOVA detected significant differences among the five genotypes, the significant differences were due to cim10 differing significantly from one or more of the other genotypes.

While evaluating adult whitefly feeding behaviors, EPG classical and three new types of E2 waveforms were identified. All four E2 waveforms were associated with the proxy for ingestion (honeydew production). Interestingly, the second type of E2 waveform, which resembled the E1 (salivation) waveform, was also associated with honeydew production. Finally, whiteflies ingesting phloem sap occasionally entered pathway phase for a brief period, and re-inserted stylets into the same sieve element to reinitiate E2, without an E1 waveform indicating that E1 was not always a prerequisite for E2.

Whitefly nymphs generated two phloem-phase waveforms (H and L) in Arabidopsis. Only waveform H was correlated with honeydew production. When nymph feeding behavior was compared among the four defense mutants or between pooled data of fast- and slow-nymphal development genotypes, no differences were found. Collectively these data indicated that phloem sap quality was the reason for differences in nymphal developmental rate.

Historically, peptidases have been considered as housekeeping proteins involved in protein degradation and amino acid turnover. However, recent work has highlighted the fact that peptidases, including aminopeptidases, have critical regulatory roles in the cell including modulation of growth, development and stress responses. Recently, plant leucine aminopeptidases (LAPs) have been recognized for their roles in modulation of late wound-responses and insect defense. While no known mechanism of action has been identified, LAPs were presumed to act through their aminopeptidase function to affect the stability, activity, or localization of a peptide and protein involved in defense. In my dissertation studies, I have identified two new molecular functions for plant LAPs as molecular chaperones and Cys-Gly dipeptidases. In addition, microarray analyses has demonstrated the tomato LAP-A regulates early and late wound responses both positively and negatively. In particular, microarray analyses identified two new sets of genes modulated by LAP-A: late wound dehydrins and Pathogenesis-Related 1. This study also provides evidence LAP-A may act though the negative regulator salicylic acid or the positive regulator hydrogen peroxide to modulate wound signaling. The role of LAP-A in glutathione metabolism is also discussed. Together this study has provided evidence that LAP-A's role in wound signaling and insect defense is more complex than initially anticipated and may be the result of LAP-A utilizing multiple functions (aminopeptidase, chaperone and/or Cys-Gly dipeptidase activities) in different environments and in response to different stresses.

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