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Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2021)

Fruit infections by fungal pathogens significantly impact the quality, marketability, and safety of produce in preharvest and postharvest scenarios. Fruit ripening, which transforms the fruit into an attractive, nutritious, and delicious food for consumers, also dramatically increases susceptibility to fungal disease. Ripening is an exceptionally complex collection of physiological and biochemical changes produced by large-scale transcriptional reprogramming in a relatively short period time, and identifying individual components of ripening that impact susceptibility is a major challenge. Furthermore, the infection strategies of necrotrophic pathogens are diverse and adapted to the ripening stage of the host.

The major objective of this Ph.D. dissertation has been to isolate specific elements of fruit ripening and fruit-pathogen interactions that have a disproportionate impact on disease outcome and which may serve as targets for breeding or postharvest control. I hypothesized that ripening-associated susceptibility is driven by accumulation of susceptibility factors (such as cell wall-degrading enzymes), and that pathogens can respond to and even manipulate these features during infection. Given the complexity of ripening and fruit-pathogen interactions, I have employed a systems-level approach, leveraging multiple multiple techniques including disease development assays, physiological assessments of ripening, measurement of phytohormones, mutant studies, cell wall glycomics, and various functional transcriptomic methods. The bulk of this research has been conducted using infections of tomato (Solanum lycopersicum) by the model necrotroph Botrytis cinerea, or gray mold (Chapters 1, 2, and 3). However, I have also used the principles and techniques developed in the tomato-B. cinerea pathosystem to expand our knowledge of fruit-pathogen interactions to other systems, specifically infections of tomato by two other necrotrophic pathogens, Rhizopus stolonifer and Fusarium acuminatum (Chapters 1 and 2), and the agriculturally important pathosystem of nectarine and Monilinia laxa, or brown rot (Chapter 4). Through this research, I have detailed infection strategies and virulence factors (Chapters 1, 3, and 4), characterized fruit responses at different developmental stages (Chapters 2, 3, and 4), and identified susceptibility factors that increase during ripening in tomato (Chapter 3).

A major emerging theme of this research has been the importance of pectin integrity and degradation in the fruit cell wall to the infection success. Both host and pathogen catabolism of pectin influence susceptibility to disease. Expression of fungal pectin-degrading enzymes is emphasized on unripe fruit, where the cell wall is more intact than it is in ripe fruit (Chapter 1). The massive upregulation of the tomato gene pectate lyase during ripening, which facilitates fruit softening, substantially increases susceptibility to B. cinerea, as demonstrated by CRISPR mutants (Chapter 2). Moreover, B. cinerea can induce the expression of multiple host pectin-degrading enzymes during infections of unripe fruit, effectively recruiting these enzymes to accelerate softening and increase susceptibility (Chapter 3). This induction appears to be dependent on the B. cinerea pectin-degrading enzymes BcPG1 and BcPG2, which may be required for the initial establishment of infection on unripe fruit (Chapter 3).

The role of the phytohormone ethylene in fruit-pathogen interactions has also obtained additional clarity through this research. Ethylene has contradictory functions in fruit-pathogen interactions, as it both promotes ripening and participates in plant defense signaling. High levels of ethylene in ripe tomato fruit and the inoculated unripe fruit of a hypersusceptible tomato mutant likely result in a net increase susceptibility, regardless of any defense signaling gained, though low, well-regulated levels of ethylene may still be useful during defense of unripe fruit up to 3 days post inoculation (Chapter 2). The eventual accumulation of increasing amounts of ethylene in response to B. cinerea after 4 days post-inoculation may be the trigger for induction of host cell wall degradation and other susceptibility-promoting ripening processes (Chapter 3). Comparable findings from gene expression analyses and ethylene measurements in the nectarine-M. laxa pathosystem indicate that this dual functionality of ethylene is similar elsewhere (Chapter 4).

Untangling increased susceptibility from fruit ripening is of tremendous importance in the ongoing battle against postharvest food losses. My research has progressed our understanding of how specific aspects of pathogen strategy, host response, and transcriptional and physiological changes during fruit ripening impact fungal disease. Additionally, the systems-level approaches I have deployed in the tomato-B. cinerea may serve as guidance for research in non-model pathosystems, as demonstrated in the study of the nectarine-M. laxa pathosystem. Ultimately, this research will assist in the management of postharvest diseases in fruit crops and facilitate breeding efforts for increased resistance to fungal pathogens in ripe fruit.

  • 2 supplemental ZIPs
Cover page: Untangling the Connections Between Fruit Ripening and Susceptibility to Fungal Pathogens
  • Article
  • Peer Reviewed
UC Davis Previously Published Works (2021)
Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.
    Cover page: Single and Double Mutations in Tomato Ripening Transcription Factors Have Distinct Effects on Fruit Development and Quality Traits
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2019)
    Worldwide, 20-25% of all harvested fruit and vegetables are lost annually in the field and throughout the postharvest supply chain due to rotting by fungal pathogens. Most postharvest pathogens exhibit necrotrophic or saprotrophic lifestyles, resulting in decomposition of the host tissues and loss of marketable commodities. Necrotrophic fungi can readily infect ripe fruit leading to the rapid establishment of disease symptoms. However, these pathogens generally fail to infect unripe fruit or remain quiescent until host conditions stimulate a successful infection. Previous research on infections of fruit has mainly been focused on the host's genetic and physicochemical factors that inhibit or promote disease. Here, we investigated if fruit pathogens can modify their own infection strategies in response to the ripening stage of the host. To test this hypothesis, we profiled global gene expression of three fungal pathogens that display necrotrophic behavior-Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer-during interactions with unripe and ripe tomato fruit. We assembled and functionally annotated the transcriptomes of F. acuminatum and R. stolonifer as no genomic resources were available. Then, we conducted differential gene expression analysis to compare each pathogen during inoculations versus in vitro conditions. Through characterizing patterns of overrepresented pathogenicity and virulence functions (e.g., phytotoxin production, cell wall degradation, and proteolysis) among the differentially expressed genes, we were able to determine shared strategies among the three fungi during infections of compatible (ripe) and incompatible (unripe) fruit tissues. Though each pathogen's strategy differed in the details, interactions with unripe fruit were commonly characterized by an emphasis on the degradation of cell wall components, particularly pectin, while colonization of ripe fruit featured more heavily redox processes, proteolysis, metabolism of simple sugars, and chitin biosynthesis. Furthermore, we determined that the three fungi were unable to infect fruit from the non-ripening (nor) tomato mutant, confirming that to cause disease, these pathogens require the host tissues to undergo specific ripening processes. By enabling a better understanding of fungal necrotrophic infection strategies, we move closer to generating accurate models of fruit diseases and the development of early detection tools and effective management strategies.
      Cover page: Infection Strategies Deployed by Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer as a Function of Tomato Fruit Ripening Stage.
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14-24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen's survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot.
        Cover page: Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen’s infection strategies
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2021)
        The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical processes associated with defense and susceptibility in tomato (Solanum lycopersicum) fruit. Using unripe and ripe fruit inoculated with three fungal pathogens, we identified common pathogen responses reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. We established that the magnitude and diversity of defense responses do not significantly impact the interaction outcome, as susceptible ripe fruit mounted a strong immune response to pathogen infection. Then, to distinguish features of ripening that may be responsible for susceptibility, we utilized non-ripening tomato mutants that displayed different susceptibility patterns to fungal infection. Based on transcriptional and hormone profiling, susceptible tomato genotypes had losses in the maintenance of cellular redox homeostasis, while jasmonic acid accumulation and signaling coincided with defense activation in resistant fruit. We identified and validated a susceptibility factor, pectate lyase (PL). CRISPR-based knockouts of PL, but not polygalacturonase (PG2a), reduced susceptibility of ripe fruit by >50%. This study suggests that targeting specific genes that promote susceptibility is a viable strategy to improve the resistance of tomato fruit against fungal disease.
          Cover page: Host susceptibility factors render ripe tomato fruit vulnerable to fungal disease despite active immune responses
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2019)
          Worldwide, 20-25% of all harvested fruit and vegetables are lost annually in the field and throughout the postharvest supply chain due to rotting by fungal pathogens. Most postharvest pathogens exhibit necrotrophic or saprotrophic lifestyles, resulting in decomposition of the host tissues and loss of marketable commodities. Necrotrophic fungi can readily infect ripe fruit leading to the rapid establishment of disease symptoms. However, these pathogens generally fail to infect unripe fruit or remain quiescent until host conditions stimulate a successful infection. Previous research on infections of fruit has mainly been focused on the host's genetic and physicochemical factors that inhibit or promote disease. Here, we investigated if fruit pathogens can modify their own infection strategies in response to the ripening stage of the host. To test this hypothesis, we profiled global gene expression of three fungal pathogens that display necrotrophic behavior-Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer-during interactions with unripe and ripe tomato fruit. We assembled and functionally annotated the transcriptomes of F. acuminatum and R. stolonifer as no genomic resources were available. Then, we conducted differential gene expression analysis to compare each pathogen during inoculations versus in vitro conditions. Through characterizing patterns of overrepresented pathogenicity and virulence functions (e.g., phytotoxin production, cell wall degradation, and proteolysis) among the differentially expressed genes, we were able to determine shared strategies among the three fungi during infections of compatible (ripe) and incompatible (unripe) fruit tissues. Though each pathogen's strategy differed in the details, interactions with unripe fruit were commonly characterized by an emphasis on the degradation of cell wall components, particularly pectin, while colonization of ripe fruit featured more heavily redox processes, proteolysis, metabolism of simple sugars, and chitin biosynthesis. Furthermore, we determined that the three fungi were unable to infect fruit from the non-ripening (nor) tomato mutant, confirming that to cause disease, these pathogens require the host tissues to undergo specific ripening processes. By enabling a better understanding of fungal necrotrophic infection strategies, we move closer to generating accurate models of fruit diseases and the development of early detection tools and effective management strategies.
            Cover page: Infection Strategies Deployed by Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer as a Function of Tomato Fruit Ripening StageCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2023)
            Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.
              Cover page: Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit
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