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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2017)

The plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) is essential for plant defense in response to biotrophic and hemibiotrophic microbial pathogens. The concentration of SA increases during infection, determining the extent of defense gene induction and cellular responses to the pathogen. In Arabidopsis thaliana, plants with non-functional avrPphB Susceptible 3 (PBS3) fail to accumulate significant induced SA and consequently lack induction of associated defense genes such as pathogenesis related 1 (PR1). These plants have increased susceptibility to pathogens such as the bacterial pathogen Pseudomonas syringae. PBS3 is a member of the GH3 family of enzymes, which conjugate small acyl substrates to amino acids. PBS3 conjugates 4-substituted hydroxybenzoic acids preferentially to glutamic acid, but this activity does not clarify its function in defense. Metabolic analyses showed that pbs3 mutants accumulate the product of another GH3 family enzyme, SA conjugated to aspartic acid (SA-Asp).

The kinetics of the known SA-Asp synthetase, GH3.5, were investigated to better understand the formation of SA-Asp in vitro. GH3.5 is also active on the growth hormone indole-3-acetic acid (IAA) and has a higher affinity for IAA than SA under moderate to high concentrations of Asp. However, when the concentration of Asp decreased, the affinity of GH3.5 for SA increased. The concentration of Asp decreases in response to pathogens, likely as part of nitrogen reallocation during the transition from growth to defense. This suggests that acyl substrate preference amongst these promiscuous enzymes can be affected by the amino acid substrate, and that GH3.5 affinity for SA is greatest during pathogen challenge.

The production of SA-Asp could serve to pull SA away from the pool used for defense in pbs3 mutants. Therefore, a pbs3gh3.5 double mutant line was created to see if the elimination of SA-Asp restored defense responses in the pbs3 background. SA-Asp was not significantly reduced in this line, so a multiplexed knockout line of likely SA-Asp synthetases was created to reduce genetic redundancy. This pbs3gh3.1gh3.3gh3.4gh3.5gh3.6 line, named gh6x, did eliminate induced SA-Asp in pbs3. However, gh6x failed to restore SA accumulation and pathogen resistance.

A genetic suppressor screen was used to identify new components in PBS3-mediated defense. Over 5,000 M2 lines were screened and ultimately two lines out of six with restored SA accumulation were chosen for further characterization. To this point, candidate causal mutations PAD4S135F and RAP2.6A93V have been identified for these two lines. PAD4 is a well-known regulator of SA-induced defense responses but may also be involved in cross talk with the SA antagonist jasmonic acid (JA). RAP2.6 is a transcription factor associated with JA and ethylene responses. The identification of these genes as candidates suggests that PBS3 may have SA-independent roles as well. RNA-sequencing identified de-repression of many JA genes in induced pbs3 as compared to induced Col-0. Furthermore, exogenous application of SA failed to restore wild type susceptibility to Pseudomonas syringae in pbs3 mutants. Taken together, these data suggest that PBS3 is important not just for the accumulation of SA, but as a higher order regulator of the complex cross talk between the mutually antagonistic SA and necrotroph-induced jasmonic acid signaling pathways.

    Cover page: Identification and characterization of genetic and molecular components of Arabidopsis thaliana PBS3-mediated salicylic acid induction during defense against microbial pathogens
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2017)
    The GH3 family of adenylating enzymes conjugate acyl substrates such as the growth hormone indole-3-acetic acid (IAA) to amino acids via a two-step reaction of acyl substrate adenylation followed by amino acid conjugation. Arabidopsis thaliana GH3.5 was previously shown to be unusual in that it could adenylate both IAA and the defense hormone salicylic acid (SA, 2-hydroxybenzoate). Our detailed studies of the kinetics of GH3.5 on a variety of auxin and benzoate substrates provides insight into the acyl preference and reaction mechanism of GH3.5. For example, we found GH3.5 activity on substituted benzoates is not defined by the substitution position as it is for GH3.12/PBS3. Most importantly, we show that GH3.5 strongly prefers Asp as the amino acid conjugate and that the concentration of Asp dictates the functional activity of GH3.5 on IAA vs. SA. Not only is Asp used in amino acid biosynthesis, but it also plays an important role in nitrogen mobilization and in the production of downstream metabolites, including pipecolic acid which propagates defense systemically. During active growth, [IAA] and [Asp] are high and the catalytic efficiency (kcat/Km) of GH3.5 for IAA is 360-fold higher than with SA. GH3.5 is expressed under these conditions and conversion of IAA to inactive IAA-Asp would provide fine spatial and temporal control over local auxin developmental responses. By contrast, [SA] is dramatically elevated in response to (hemi)-biotrophic pathogens which also induce GH3.5 expression. Under these conditions, [Asp] is low and GH3.5 has equal affinity (Km) for SA and IAA with similar catalytic efficiencies. However, the concentration of IAA tends to be very low, well below the Km for IAA. Therefore, GH3.5 catalyzed formation of SA-Asp would occur, fine-tuning localized defensive responses through conversion of active free SA to SA-Asp. Taken together, we show how GH3.5, with dual activity on IAA and SA, can integrate cellular metabolic status via Asp to provide fine control of growth vs. defense outcomes and hormone homeostasis.
      Cover page: Preference of Arabidopsis thaliana GH3.5 acyl amido synthetase for growth versus defense hormone acyl substrates is dictated by concentration of amino acid substrate aspartate
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2021)
      Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.
        Cover page: Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance
        • Article
        • Peer Reviewed
        Biological Systems & Engineering (2022)
        The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.
          Cover page: Making Security Viral: Shifting Engineering Biology Culture and Publishing
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