Interactions between plant defense and prior herbivory alter conditions for a plant virus and its vector

Background: While virus-vector-host interactions have been a major focus of both basic and applied ecological research, little is known about how different levels of plant defense interact with prior herbivory to affect these relationships. We used genetically-modified strains of tomato (Solanum lycopersicum) varying in the jasmonic acid (JA) plant defense pathways to explore how plant defense and prior herbivory affects a plant virus (tomato yellow leaf curl virus, 'TYLCV'), its vector (the whitefly Bemisia tabaci MED), and the host. Results: Virus-free MED preferred low-JA over high-JA plants and had lower fitness on high-JA plants. Viruliferous MED preferred low-JA plants but their survival was unaffected by JA levels. While virus-free MED did not lower plant JA levels, viruliferous MED decreased both JA levels and the expression of JA-related genes. Infestation by viruliferous MED reduced plant JA levels, and TYLCV loads were consistently higher in plants fed upon by viruliferous MED than in plants fed on by virus-free MED. In preference tests, neither virus-free nor viruliferous MED discriminated among JA-varying plants previously exposed to virus-free MED. However, both virus-free and viruliferous MED preferred low-JA plant genotypes when choosing between plants that had both been previously exposed to viruliferous MED. The enhanced preference for low-JA genotypes appears linked to the volatile compound neophytadiene, which was found only in whitefly-infested plants and at concentrations inversely related to plant JA levels. Conclusions: Our findings illustrate how plant defense can interact with prior herbivory to affect both a plant virus and its whitefly vector, and confirm the induction of neophytadiene by lower JA that may prove useful in pest detection and management. interaction, the molecular mechanisms

JA levels were lower in spr2 versus 35S plant genotypes, and in plants exposed to viruliferous MED (Fig. 2E). While virus-free MED did not reduce JA levels in any of the three plant genotypes, viruliferous MED reduced JA by 85% in WT plants and 77% in 35S plants (p < 0.05 for both; Table 1C). Viruliferous MED did not reduce JA in spr2 plants.
Virus-free and viruliferous MED did not affect the expression of JA-related genes in spr2 plants. There was no effect of virus-free MED on the expression of JA-related genes in 35S plants.
Neophytadiene was the only compound exclusively associated with whitefly infestation. It was found only in plants fed upon by viruliferous MED, in concentrations that were negatively correlated with plant JA levels (i.e., spr2 > WT > 35S plants, Fig. 4). Other than neophytadiene, there were no compounds consistently (i.e., in two or more plant genotypes) associated with viruliferous MED. Prior infestation by virus-free MED, however, increased β-phellandrene concentrations in all three plant genotypes (all p < 0.05), and 1hexanol concentrations in spr2 and 35S plants (Fig. 4A,C).
Neither virus-free nor viruliferous MED differentiated between plant genotypes that had previously been fed upon by virus-free MED (p > 0.05 for all; Fig. 5A,B). In contrast, both virus-free and viruliferous MED strongly preferred lower-JA genotypes when choosing between two plant genotypes that had both been infested by viruliferous MED (p < 0.05 for all; Fig. 5C,D).

Discussion
Our findings agree with work showing that JA pathways provide an effective defense against phloem-feeding herbivores. Both aphids and B. tabaci MEAM1 , for instance, develop more quickly on JA-impaired versus JA-enhanced Arabidopsis thaliana genotypes (21,22). We also found that both viruliferous MEAM1 and MED had decreased fecundity, longevity, and survival rates in JA-treated plants compared with control plants (Shi et al. 2017). However, here we found that JA-based defense did not affect TYLCV-carrying MED: while virus-free MED grew more slowly on higher-JA plants, viruliferous MED were unaffected by feeding on these plant genotypes (Fig. 1). This may result from differences between control plants treated with 1mM JA spray and high JA transgenic plants; the inhibitory effect of 1 mM JA may be much higher than that present in the JA transgenic plants.
Feeding by virus-free MED increased expression of the JA-biosynthesis-related LOX and OPR3 genes, although there was no corresponding increase in either JA levels or expression of the JA-induced genes PI II and JAR1 (Fig. 2). These results contrast with previous research showing that virus-free MEAM1 suppresses herbivore-induced JA compared with undamaged controls (23,24). The difference between our results and prior work may results from differences between tomato varieties used in the research and/or genotype difference between MEAM1 and MED. In contrast, viruliferous MED both decreased the expression of JA-related genes ( Fig thaliana (25), for example, and co-infection of the begomovirus TYLCCNV and its betasatellite suppressed JA-based defense against MEAM1 in tobacco (16). While both TYLCCNV and its beta-satellite are required to effectively suppress host defense, TYLCV is a true monopartite begomovirus that lacks a beta-satellite (26). Our results thus demonstrate that the monopartite begomovirus TYLCV can suppress even constitutively-expressed plant defenses and increase both vector and virus fitness. The impact of plant defenses on this suppression is shown by the fact that supported by the fact that although viruliferous MED reduced JA levels in all three plant genotypes, TYLCV levels were still lower in 35S plants than the other two genotypes (Fig. 3A, B). Lower viral titers in the JA-overexpressing line agree with work suggesting that JA can slow viral replication (5); activation of this defense pathway in Phaseolus vulgaris, for example,inhibited white clover mosaic potex virus (27).
When choosing between uninfested plants, both virus-free and viruliferous MED exhibited at least a marginal preference for the low-JA spr2 over the high-JA 35S genotype but did not differentiate between WT plants and either genotype. The preference for low-JA genotypes was stronger when whiteflies chose between plants that had both been fed upon by viruliferous MED (Fig. 5). The strongest preference of both virus-free and viruliferous MED was for spr2 over 35S (74% preference), but their low-JA preference was still significant when choosing between the JA-varying genotypes and WT plants. The from Verbascum thapsus, a plant high in neophytadiene, have also been found to attract whiteflies (29). Four other terpenes, α-phellandrene, (+)-2-carene, β-caryophyllene, and αhumulene were induced by infestations of virus-free MED but reduced by viruliferous MED.
All four compounds have been reported to exhibit repellent properties to whiteflies ( 30,31), and their reduced expression in viruliferous MED plants likely reflects virally-induced reduction of plant defense.
By addressing how plant defense and its interaction with prior herbivory affects the hostvector-virus interaction, our results suggest several avenues for additional research. The ability of herbivorous insects to suppress host defenses and manipulate plant volatile emissions, for instance, often involves feeding-transmitted symbionts or compounds (32).  Footnote: "df" refers to "degree of freedom"; " F" refers to "F value" ; "p" refers to "p    Asterisk indicates a significant difference in the three genotypes (P < 0.05).