Mitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown. Here, we show a role for the GTPase Miro2 in mitochondria interaction with the ER and its impacts on mitochondria fusion and motility. Mutations in AtMiro2's GTPase domain indicate that the active variant results in larger, fewer mitochondria which are attached more readily to the ER when compared with the inactive variant. These results are contrary to those in metazoans where Miro predominantly controls mitochondrial motility, with additional GTPases affecting fusion. Synthetically controlling mitochondrial fusion rates could fundamentally change plant physiology by altering the energy status of the cell. Furthermore, altering tethering to the ER could have profound effects on subcellular communication through altering the exchange required for pathogen defence.

In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress crossprotection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack. HighlightsFungal pathogens have evolved anticipatory behaviors that protect against imminent challenges in the host, such as immune attack.

Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their “pathogen-associated molecular patterns” (PAMPs), which are recognized as “foreign” and used to activate protective immunity.

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.

Most microbes have developed responses that protect them against stresses relevant to their niches. Some that inhabit reasonably predictable environments have evolved anticipatory responses that protect against impending stresses that are likely to be encountered in their niches–termed “adaptive prediction”. Unlike yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis and Yarrowia lipolytica and other pathogenic Candida species we examined, the major fungal pathogen of humans, Candida albicans, activates an oxidative stress response following exposure to physiological glucose levels before an oxidative stress is even encountered. Why? Using competition assays with isogenic barcoded strains, we show that “glucose-enhanced oxidative stress resistance” phenotype enhances the fitness of C. albicans during neutrophil attack and during systemic infection in mice. This anticipatory response is dependent on glucose signalling rather than glucose metabolism. Our analysis of C. albicans signalling mutants reveals that the phenotype is not dependent on the sugar receptor repressor pathway, but is modulated by the glucose repression pathway and down-regulated by the cyclic AMP-protein kinase A pathway. Changes in catalase or glutathione levels do not correlate with the phenotype, but resistance to hydrogen peroxide is dependent on glucose-enhanced trehalose accumulation. The data suggest that the evolution of this anticipatory response has involved the recruitment of conserved signalling pathways and downstream cellular responses, and that this phenotype protects C. albicans from innate immune killing, thereby promoting the fitness of C. albicans in host niches.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis. Although certain virulence factors have been proposed as potential sporotrichosis determinants, the scarcity of molecular tools for reverse genetics studies on Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous expression in S. brasiliensis, S. schenckii and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol allowed the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homologue that is critical for non-homologous end-joining DNA repair. The use of S. brasiliensis delta ku80 strains enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species.

S9.4 Free oral presentations (late breaking), September 23, 2022, 4:45 PM - 6:15 PM Candida albicans adaptation to host niches affects the exposure of key pathogen-associated molecular patterns (PAMPs) on its cell surface and, consequently, the detection of C. albicans cells by the immune system. Focusing on β-(1,3)-glucan, we screened for host inputs that influence the exposure of this immune-stimulatory PAMP on the C. albicans cell surface. We used a combination of fluorescent microscopy, flow cytometry, and cytokine assays, and then analyzed certain conditions in more detail using transmission electron microscopy and time-lapse video microscopy of C. albicans-phagocyte interactions. We found that some nutrients, micronutrient limitation, stresses, and antifungal drugs trigger β-glucan masking, whereas other inputs, such as nitrogen sources and quorum sensing molecules, exert limited effects on β-glucan exposure. In particular, host- or bacterial-derived L-lactate, hypoxia, or iron limitation induce β-glucan masking, and this leads to attenuation of phagocytic responses [Nature Micro 2, 16 238; mBio 9, e01318-18; Nature Comms 10, 5315]. Lactate signals through Gpr1 to activate Crz1 in a calcineurin-independent manner, whereas hypoxia signals via mitochondrial ROS, and iron limitation signals through Ftr1 and Sef1. β-glucan masking also depends upon downstream signaling via the cAMP-PKA pathway. We conclude that C. albicans has evolved to exploit a range of specific host-derived signals to modulate the exposure of a major PAMP at its cell surface in an attempt to evade phagocytic uptake. Using barcode-sequencing in direct competition assays in vivo, we showed that preadaptation to specific β-glucan masking signals affects the ability of this fungus to colonize particular tissues during systemic infection in a murine model. This reinforces the view that β-glucan masking promotes C. albicans infection.

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.