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.

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.