The biochemical basis of plant ATG8 substrate specificity

Autophagy is an essential eukaryotic cellular quality control pathway that involves the degradation of self- and non-self macromolecules , with multiple layers of specificity defining the dynamics of substrate uptake, sub-cellular trafficking, and turnover. ATG8 is a highly-conserved ubiquitin-like protein that is central to the selectivity of the autophagy pathway, directly or indirectly binding desired autophagic cargo. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins is unclear. In this thesis, I described the potato ATG8 interactome using in planta immunoprecipitation followed by mass spectrometry, discovering that potato ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. In addition, I defined the biochemical basis of potato ATG8 specialization. I revealed that the ATG8 N-terminal β-strand underpins binding specificity to substrates that contain ATG8-interacting motifs (AIMs), including the ATG8targetting effector from the potato late blight pathogen Phytophthora infestans, PexRD54. To approach the question of ATG8 substrate specificity from the opposing direction, I also explored the evolutionary dynamics of PexRD54 in different host-specific lineages of Phytophthora. I found that the PexRD54 ortholog from P. mirabilis, a closely related species to P. infestans, has a polymorphism in its AIM which nearly abolishes binding to the ATG8s of its host, Mirabilis jalapa. These results provide insights into the requirements of a functional ATG8-interacting motif, as well as raise questions as to whether specific selective pressures of the M. jalapa host environment have shaped the evolution the P. mirabilis PexRD54

The coming of age of EvoMPMI:evolutionary molecular plant-microbe interactions across multiple timescales

Plant-microbe interactions are great model systems to study co-evolutionary dynamics across multiple timescales. However, mechanistic research on plant-microbe interactions has often been conducted with little consideration of evolutionary concepts and methods. Conversely, evolutionary research has rarely integrated the range of mechanisms and models from the molecular plant-microbe interactions field. In recent years, the incipient field of evolutionary molecular plant-microbe interactions (EvoMPMI) has emerged to bridge this gap. Here, we report on some of the recent advances in EvoMPMI. In particular, we highlight new systems to study microbe interactions with early diverging land plants, and new findings from studies of adaptive evolution in pathogens and plants. By linking mechanistic and evolutionary research, EvoMPMI promises to expand our understanding of plant-microbe interactions

Regressive evolution of an effector following a host jump in the Irish potato famine pathogen lineage

In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution of an effector activity in the Irish potato famine pathogen (Phytophthora infestans) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across Phytophthora species. We found that PexRD54 emerged in the common ancestor of Phytophthora clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the P. mirabilis PexRD54 (PmPexRD54) AIM is atypical, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the Mirabilis jalapa host environment. We began by characterizing the M. jalapa ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind weakly to the M. jalapa ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conclude that the functionality of the PexRD54 AIM was lost in the P. mirabilis lineage, perhaps owing to as-yet-unknown selection pressure on this effector in the new host environment

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants

An organic acid based counter selection system for cyanobacteria.

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803)

Specific activity of AcsA towards the organic acids acetate, propionate, acrylate, and 3HP.

<p>Specific activity of AcsA towards the organic acids acetate, propionate, acrylate, and 3HP.</p

Comparison of <i>acsA</i> and <i>glpK</i> mutant phenotypes.

<p>(A) Growth of wild type PCC 7002 (green diamonds) and BPSyn_014 (blue squares) in Medium A⁺ supplemented with air. (B) Fluorescence measurements of strains BPSyn_015 and BPSyn_027, which contain YFP under the <i>cpcB</i> promoter in the <i>acsA</i> or <i>glpK</i> locus, respectively. Fluorescence is measured using arbitrary units (A.U.) at an excitation of 514 nm and an emission of 527 nm. Data points are the mean of biological triplicates and error bars represent the standard deviation.</p

Homozygous mutants were obtained in both the <i>acsA</i> and <i>glpK</i> loci.

<p>(A) Diagram of the primers used to screen for homozygous strains in the <i>acsA</i> locus. Primer set A amplifies from the up and down5' and 3' stream flanking regions of <i>acsA</i> and should be amplify 3.2 kb, 1.1 kb, and 2.6 kb in fragments for WT 7002, BPSyn_014, and BPSyn_015, respectively. Primer set B amplifies 1.2 kb from inside <i>acsA</i> to the region downstream of <i>acsA</i>. Primer set C amplifies 0.9 kb from the barcode (BC) region to downstream of <i>acsA</i>. Primer set D amplifies a 0.25 kb region internal to YFP. (B) 1% agarose gel showing colony PCR products amplified using the primer sets A, B, C, and D on WT PCC 7002, BPSyn_014, and BPSyn_015. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets A, B, C, and D amplifying WT PCC 7002, BPSyn_014, and BPSyn_015, respectively. (C) Diagram of the primers used to screen for homozygous strains in the <i>glpK</i> locus. Primer set E amplifies from the up and down stream5' and 3' flanking regions of <i>glpK</i> and should be amplify 2.7 kb, 4.8 kb, and 2.6 kb fragments from BPSyn_014, BPSyn_022, and BPSyn_027, respectively. Primer set F amplifies 0.72 kb from inside <i>glpK</i> to the region downstream of <i>glpK</i>. Primer set G amplifies a 2.3 kb region of the <i>acsA</i> cassette that was introduced into the <i>glpK</i> locus. (D) 1% agarose gel showing colony PCR products amplified using the primer sets E, F, G, and D on BPSyn_014, BPSyn_022, and BPSyn_027. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets E, F, G, and D amplifying BPSyn_014, BPSyn_022, and BPSyn_027, respectively.</p

Outline of counter selection protocol based on acrylate sensitivity.

<p>Outline of counter selection protocol based on acrylate sensitivity.</p

Growth of wild type and an adapted strain of PCC 7002 with acrylate.

<p>(A) Growth of wild type PCC 7002 in unmodified medium (green diamonds) or 5 mM acrylate (red squares). (B) Growth of acrylate adapted cultures of PCC 7002 in unmodified medium (green diamonds) or 5 mM acrylate (red squares). Data points are the mean of biological triplicates and error bars represent the standard deviation.</p