Many of the genes in the MG core response were either functionally similar to other RR core response genes or were expressed at low levels. Thus, the ability to mount an immune response does not appear to be compromised in RR fruit. If defense responses do not determine the outcome of the interaction in tomato fruit, developmental features associated with ripening of healthy fruit may instead govern susceptibility. The highly complex transcriptional reprogramming during ripening allows for a large number of potential contributors to the increase in susceptibility. Ripening processes in tomato have been studied using non-ripening mutants such as Cnr, rin, and nor. In addition to being phenotypically distinct, these mutants display differential susceptibility patterns when inoculated with fungal pathogens . Previously, susceptibility to B. cinerea in tomato fruit was shown to be dependent on NOR but not RIN, though the role of CNR remained uncharacterized . Our results with B. cinerea as well as F. acuminatum and R. stolonifer corroborate the roles of NOR and RIN while also proposing a role for CNR in tomato fruit defense against fungal pathogens. In addition to exhibiting hypersusceptibility to B. cinerea in RR-like fruit, Cnr MG-like fruit were the only fruit of this stage to exhibit any susceptibility. Unlike rin and nor fruit, Cnr fruit have altered cell wall architecture even in MG-like stages , a feature which may be exploited during fungal infection. Moreover, large plastic pots compared with all other fruit, Cnr MG-like fruit were deficient in defense responsesagainst B. cinerea.
Apart from Cnr MG-like fruit, the extent of the immune responses appeared to have little impact on susceptibility, as enriched defense categories were similar across both resistant and susceptible mutant fruit. We took advantage of the susceptibility differences in the ripening mutants to unravel ripening components that may represent either declining preformed defenses or increasing susceptibility factors. Differential expression analyses carefully filtered based on susceptibility phenotypes revealed that several defense-related genes undergo changes in gene expression during the transition from MG/MG-like to RR/RR-like fruit. Most interestingly, declining preformed defenses appear to be over-represented by gene categories involved in the mediation of ROS levels. Host regulation of ROS levels during early fungal infection is critical for both defense signaling and detoxification of ROS generated by the pathogen , and tomato fruit susceptibility to B. cinerea has been shown to be impacted by both of these roles. Improved resistance to B. cinerea in the ABA deficient sitiens mutant has been shown to be the result of controlled ROS production, which promotes cell wall fortification , and a similar improved B. cinerea resistance is seen in tomato varieties genetically engineered to produce especially high amounts of antioxidant anthocyanins in fruit . During ripening, losing control of ROS levels may thus represent the reduction of an important preformed defense.
Some features of ripening have the potential to be either a preformed defense or a susceptibility factor depending on the context. Although ethylene is known for its involvement in defense against necrotrophs , its induction of the ripening program catalyses downstream events that can be favorable for pathogen infections. Previous research suggests that inhibition of ethylene receptors in MG fruit can either increase or decrease resistance to B. cinerea depending on the concentration of inhibitor used . Thus, ethylene-mediated resistance may be dependent on careful regulation of ethylene levels, and the autocatalytic ethylene biosynthesis that occurs in wild-type fruit ripening may be detrimental. We observed that ethylene production and ethylene-related transcriptional responses were particularly prominent in susceptible fruit, especially Cnr MG-like. In addition to ethylene, JA is known to mediate resistance to necrotrophs in plants . The enrichment of JA biosynthesis genes is seen in the RR core response, as well as the response to B. cinerea in all mutant fruit at both stages. Basal levels of JA in healthy fruit are highest in nor RR-like fruit, where they are nearly twice as high as levels in wild-type RR fruit. Moreover, nor fruit are the only fruit at which JA signaling/response genes are enriched in response to B. cinerea infection at both stages. The interplay between ethylene and JA and their impact of ripening-associated susceptibility requires further study. Other features of ripening can increase susceptibility to fungal disease such as the disassembly of plant cell walls leading to fruit softening.
Cell wall polysaccharide remodeling, breakdown, and solubilization in ripening fruit occurs as the result of various cell wall-degrading enzymes, particularly those that act on pectin . The cell wall represents an important physical barrier to pathogen attack in plants , and cell wall integrity and fortification improves tomato fruit resistance to B. cinerea infection . The enzymes PL and PG2a feature prominently in tomato fruit ripening and softening and accumulate in RR/RR-like fruit of susceptible genotypes. However, these enzymes do not have equal impact on fruit softening, as CRISPR-based mutants in PL, but not PG2a, result in a reduced rate of softening in RR fruit . This differential impact on firmness is mirrored in the effect on susceptibility to B. cinerea, as the firmer CRISPR-PL mutant was less susceptible than both the CRISPR-PG2a mutant and the azygous control . Though RR fruit of the CRISPR-PG2a mutant did not exhibit increased B. cinerea resistance, PG2a may still contribute to susceptibility, as RNAi-mediated knockdown of PG2a together with the expansin gene Exp1 increases B. cinerea resistance while knockdown of either gene alone does not . Here we showed that the PL enzyme is a substantial susceptibility factor in tomato fruit, and targeting this enzyme for breeding purposes may improve fungal resistance in addition to lengthening shelf life by slowing the softening process. Susceptibility and resistance to necrotrophic pathogens is ultimately a complex, multigenic trait in plants. The use of transcriptomic datasets to facilitate a systems-level approach of such pathosystems has increased in recent years and has led to novel insights in both host and pathogen features that impact the outcome of such interactions. Moreover, the additional layer of an enormous developmental change such as ripening only further increases the need for these approaches. We have demonstrated how such an approach can yield critical information on both fruit infection response and broad ripening-associated changes that increase susceptibility, and additionally provide insights into single genes with a disparate impact on susceptibility. From our results, we believe that ripening-associated susceptibility is best explained by a dominant role of susceptibility factors that increase during ripening, which, coupled with a modest loss of preformed defenses, outweighs the efforts of the immune response in ripe fruit . Overall, our results have tremendous utility for guiding future study of fruit–pathogen interactions in addition to providing breeders with information on potentially useful genes for targeting in the hopes of ultimately reducing post harvest losses in tomatoes and other fruit crops.Globalization of human trade has led to great economic advances but has also resulted in serious environmental and economic threats in the form of the transport of organisms across large geographic distances.
When a transported species ends up flourishing in its new home to the detriment of the preexisting ecosystem or to our economic interests, we term this species “invasive”. In the world of entomology, raspberry container invasion biology is often associated with many serious problems, including vectoring animal and plant diseases, agricultural crop losses, and competition with native species. Focusing on plant pests, the most significant factors in the spread of invasive pests are the international trade networks of live plants, forestry products, and seeds . Thus, in the modern era of global shipping and trade, the cost of preventing and controlling invasive pests has only grown higher, with the cost of management and damage growing threefold every decade since 1970 . An important aspect of invasion biology studies is the understanding of population history and structure. Knowledge of where an invasive species originated can allow government agencies to set up quarantine and shipping policies to prevent further introduction of the species, and prevent repeated reintroductions that increase transfer of genetic diversity to the invaded region, boosting the invasive species’ chance of success . Additionally, understanding whether distinct populations exist in an invaded area, or whether migrations are occurring between populations, can allow management efforts to customize tailored approaches to controlling each population . Historically, molecular markers such as microsatellites, amplified fragment length polymorphisms, or mitochondrial sequences were used to differentiate populations and reconstruct population or species history . However, cost reduction of whole-genome sequencing technologies have enabled relatively cheap access to hundreds of thousandsof single nucleotide polymorphism markers. Non-coding SNPs distributed across the genome are ideal for accurately representing population history, as they are assumed to be unaffected by selective pressures . As long as individuals sequenced are sampled randomly across the regions of interest, using a large number of genomic SNPs from a relatively small number of individuals can be sufficient for assessing population relationships with one another . Thus, the large increase in number of markers that can be interrogated is driving the ability to recognize increasingly complex invasion patterns . In my first chapter, I perform a population analysis of Drosophila suzukii, an invasive vinegar fly originally from Asia . D. suzukii was detected in Spain and California in 2008 , and within a few years rapidly spread worldwide. This pest is a particular problem to soft-flesh berries such as blueberries and raspberries, due to the serrated ovipositor of the female that allows it to lay eggs under the skin of fresh fruit . I used whole genome sequencing of hundreds of individual Drosophila suzukii flies to identify population structure and found evidence of two major populations in the United States, composed of an Eastern and Western group, but no evidence of North to South differentiation. We followed up this discovery with a migration analysis and found evidence that the Western U.S. populations originated from an Asian migration event with subsequent migrations from Hawaii. We additionally found evidence of back-migration from the Western U.S. to Asia, indicating that Drosophila suzukii is being exchanged in both directions, likely by agricultural shipping. Significantly, invasive populations in the U.S. or Europe showed no loss in genetic diversity relative to Asian ancestors, suggesting migrating populations are not undergoing bottlenecks or that migrations are ongoing, continually supplying genetic diversity to invaded regions . In my second chapter, I perform a similar analysis of population structure on Tuta absoluta, a moth responsible for massive tomato crop losses worldwide. T. absoluta was an agricultural pest in SouthAmerica throughout the late 20th century before it was detected in Spain in 2006 before subsequently spreading across Europe, Africa, and Asia . For this analysis I focused on understanding the population structure of T. absoluta in Latin America. I first assembled and annotated a new genome using long-read technology to improve gene annotation and variant calling. Using whole genome sequencing of individuals collected across Latin America, I found evidence for three major clusters, with the Chilean populations identified the source of the initial European invasion in 2006. Selection statistics and diversity levels show several genomic regions have experienced recent selective sweeps. Using the new genome annotations, I found these regions contain genes important to insecticide resistance, immunity, and metabolism. In addition to understanding population-level dynamics of invasive species, genomic data can also be used to develop molecular diagnostics, either for the purpose of identifying the species or identifying the geographic source of an individual . Having access to highly reliable detection methods of an invasive species is key to any eradication events, both in detecting its presence, but also in detecting its absence so that prevention efforts can be efficiently used elsewhere . While identification by morphology is typically used for insects, this strategy can become challenging if the insect is extremely small or is morphologically similar to other species already present in the surveilled area. Molecular diagnostics have the advantage of not requiring expert knowledge of the species’ traits, and can often be faster, cheaper, and more accurate . In my third chapter, I used available genomic data to develop a molecular diagnostic to identify T. absoluta. Unlike D. suzukii, T. absoluta has not yet been detected in the United States. Monitoring farms and greenhouses for the appearance of T. absoluta is a critical step in preventing the establishment of this pest, but identification is hampered by morphologically identical species already in the U.S. that inhabit the same ecological niche. Thus, I develop two types of molecular diagnostics that enabledetection of T. absoluta from DNA samples.