Sphingolipid biosynthetic pathway genes FEN1 and SUR4 modulate amphotericin B resistance.

Deletants of the sphingolipid biosynthetic pathway genes FEN1 and SUR4 of Saccharomyces cerevisiae, as well as deletants of their orthologs in Candida albicans, were found to be 2- to 5-fold-more sensitive to amphotericin B (AmB) than parent strains. The inhibition of sphingolipid biosynthesis in parent strains by myriocin sensitized them to AmB, which can be reversed by providing phytosphingosine, an intermediate in the sphingolipid pathway. These results indicate that sphingolipids modulate AmB resistance, with implications for mechanisms underlying AmB action and resistance.

Use of a visible reporter marker- myb-related gene in crop plants to minimize herbicide usage against weeds.

Weeds, a main threat to agricultural productivity worldwide, are mostly controlled by herbicides. To minimize herbicide usage by targeting only weedy areas, we developed a new methodology for robust weed detection that relies on manipulating the crop plant's leaf hue, without affecting crop fitness. We generated transgenic tobacco (Nicotiana tabacum Xanthi) lines overexpressing the anthocyanin pigment as a traceable marker that differentiates transgenes from the surrounding weeds at an early stage. Transformation with the anthocyanin VlmybA1-2 gene produced purple-colored leaves. Subsequent gene silencing with vector pTRV2:VlmybA1-2 significantly reduced anthocyanin pigments in tobacco leaves 40 days after agroinfiltration, with a concomitant reduction in VlmybA1-2 transcript levels. Purple hue faded gradually, and there were no fitness costs in terms of plant height or leaf number in the silenced vs. non-silenced tobacco transgenes. These results could lead to a new sustainable weed-control method that will alleviate weed-related ecological, agricultural and economic issues.

Critical role for CaFEN1 and CaFEN12 of Candida albicans in cell wall integrity and biofilm formation.

Sphingolipids are involved in several cellular functions, including maintenance of cell wall integrity. To gain insight into the role of individual genes of sphingolipid biosynthetic pathway, we have screened Saccharomyces cerevisiae strains deleted in these genes for sensitivity to cell wall perturbing agents calcofluor white and congo red. Only deletants of FEN1 and SUR4 genes were found to be sensitive to both these agents. Candida albicans strains deleted in their orthologs, CaFEN1 and CaFEN12, respectively, also showed comparable phenotypes, and a strain deleted for both these genes was extremely sensitive to cell wall perturbing agents. Deletion of these genes was reported earlier to sensitise cells to amphotericin B (AmB), which is a polyene drug that kills the cells mainly by binding and sequestering ergosterol from the plasma membrane. Here we show that their AmB sensitivity is likely due to their cell wall defect. Further, we show that double deletant of C. albicans is defective in hyphae formation as well as biofilm development. Together this study reveals that deletion of FEN1 and SUR4 orthologs of C. albicans leads to impaired cell wall integrity and biofilm formation, which in turn sensitise cells to AmB.

Increased LOH due to Defective Sister Chromatid Cohesion Is due Primarily to Chromosomal Aneuploidy and not Recombination.

Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.

Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway.

Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3.