A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress.

The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii, we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a ß-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress.This article has an associated First Person interview with the first author of the paper.

Regulation of nitrate and methylamine metabolism by multiple nitrogen sources in the methylotrophic yeast Candida boidinii.

The methylotrophic yeast Candida boidinii, which is capable of growth on methanol as a sole carbon source, can proliferate on the leaf surface of Arabidopsis thaliana. Previously, we demonstrated that adaptation to a change in the major available nitrogen source from nitrate to methylamine during the host plant aging was crucial for yeast survival on the leaf environment. In this report, we investigated the regulatory profile of nitrate and methylamine metabolism in the presence of multiple nitrogen sources in C. boidinii. The transcript level of nitrate reductase (Ynr1) gene was induced by nitrate and nitrite, and was not repressed by the coexistence with other nitrogen sources. In contrast, the transcript level of amine oxidase (Amo1) gene, which was induced by methylamine, was significantly repressed by the coexistence with ammonium or glutamine. In addition, we investigated the intracellular dynamics of Ynr1 during the nitrogen source shift from nitrate to other compounds. Under these tested conditions, Ynr1 was effectively transported to the vacuole via selective autophagy only during the shift from nitrate to methylamine. Moreover, Ynr1 was subject to degradation after the shift from nitrate to nitrate plus methylamine medium even though nitrate was still available. These regulatory profiles may reflect life style of nitrogen utilization in this yeast living in the phyllosphere.

Autophagy as a Survival Strategy for Eukaryotic Microbes Living in the Phyllosphere.

Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding yeast, but the physiological role of autophagy in yeast cells had remained unknown as autophagy-deficient yeast mutants did now show a clear growth phenotype in laboratory conditions. In this review, we introduce the role of autophagy in the methylotrophic yeast Candida boidinii grown on the leaf surface of Arabidopsis thaliana. Autophagy is shown to be required for proliferation in the phyllosphere, and selective autophagic pathways such as pexophagy and cytoplasm-to-vacuole targeting (Cvt) pathway are strictly regulated during both the daily cycle and the host plant life cycle. This review describes our current understanding of the role of autophagy as a survival strategy for phyllosphere fungi. Critical functions of autophagy for pathogen invasions are also discussed.

Physiology of Methylotrophs Living in the Phyllosphere.

Methanol is abundant in the phyllosphere, the surface of the above-ground parts of plants, and its concentration oscillates diurnally. The phyllosphere is one of the major habitats for a group of microorganisms, the so-called methylotrophs, that utilize one-carbon (C1) compounds, such as methanol and methane, as their sole source of carbon and energy. Among phyllospheric microorganisms, methanol-utilizing methylotrophic bacteria, known as pink-pigmented facultative methylotrophs (PPFMs), are the dominant colonizers of the phyllosphere, and some of them have recently been shown to have the ability to promote plant growth and increase crop yield. In addition to PPFMs, methanol-utilizing yeasts can proliferate and survive in the phyllosphere by using unique molecular and cellular mechanisms to adapt to the stressful phyllosphere environment. This review describes our current understanding of the physiology of methylotrophic bacteria and yeasts living in the phyllosphere where they are exposed to diurnal cycles of environmental conditions.

Yeast nitrogen utilization in the phyllosphere during plant lifespan under regulation of autophagy.

Recently, microbe-plant interactions at the above-ground parts have attracted great attention. Here we describe nitrogen metabolism and regulation of autophagy in the methylotrophic yeast Candida boidinii, proliferating and surviving on the leaves of Arabidopsis thaliana. After quantitative analyses of yeast growth on the leaves of A. thaliana with the wild-type and several mutant yeast strains, we showed that on young leaves, nitrate reductase (Ynr1) was necessary for yeast proliferation, and the yeast utilized nitrate as nitrogen source. On the other hand, a newly developed methylamine sensor revealed appearance of methylamine on older leaves, and methylamine metabolism was induced in C. boidinii, and Ynr1 was subjected to degradation. Biochemical and microscopic analysis of Ynr1 in vitro during a shift of nitrogen source from nitrate to methylamine revealed that Ynr1 was transported to the vacuole being the cargo for biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway, and degraded. Our results reveal changes in the nitrogen source composition for phyllospheric yeasts during plant aging, and subsequent adaptation of the yeasts to this environmental change mediated by regulation of autophagy.