Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen’s diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

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Opportunistic fungal infections are a leading cause of death for immune-compromised patients and there is pressing need to develop new anti-fungal therapeutic agents because of toxicity and resistance to current anti-fungal drugs. Although C-type lectin receptor- and Toll-like receptor-induced signaling pathways are key activators of host anti-fungal immunity, little is known about the negative regulation of these immune responses. Here, we found that JNK1 activation suppresses anti-fungal immunity in mice. We showed that JNK1-deficient mice had significantly higher survival rate in response to Candida albicans infection, and JNK1 expressed in hematopoietic innate immune cells is critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 promoter. Blocking CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced anti-fungal effect in JNK1-deficient mice. Notably, JNK inhibitors exerted potent anti-fungal therapeutic effects in Candida albicans-infected mouse and human cells, indicating that JNK1 can be a therapeutic target for treating fungal infection.

The activation of both the SA and JA/ETsignalling pathways may lead to more efficient general and broad resistance to Pst DC3000 by non-pathogenic rhizobacteria. However, the mechanisms that govern this simultaneous activation are unclear. Using Arabidopsis as a model system, two transcription factors, WRKY11 and WRKY70, were identified as important regulators involved in Induced Systemic Resistance (ISR) triggered by Bacillus cereus AR156. The results revealed that AR156 treatment significantly stimulated the transcription of WRKY70, but suppressed that of WRKY11 in Arabidopsis leaves. Furthermore, they were shown to be required for AR156 enhancing the activation of cellular defence responses and the transcription level of the plant defence response gene. Overexpression of the two transcription factors in Arabidopsis also showed that they were essential for AR156 to elicit ISR. AR156-triggered ISR was completely abolished in the double mutant of the two transcription factors, but still partially retained in the single mutants, indicating that the regulation of the two transcription factors depend on two different pathways. The target genes of the two transcription factors and epistasis analysis suggested that WRKY11 regulated AR156-triggered ISR through activating the JA signalling pathway, and WRKY70 regulated the ISR through activating the SA signalling pathway. In addition, both WRKY11 and WRKY70 modulated AR156-triggered ISR in a NPR1-dependent manner. In conclusion, WRKY11 and WRKY70 played an important role in regulating the signalling transduction pathways involved in AR156-triggered ISR. This study is the first to illustrate the mechanism by which a single rhizobacterium elicits ISR by simultaneously activating both the SA and JA/ET signalling pathways.

Innate immune responses mediated by C-type lectin receptors Dectin-2 and Dectin-3 against fungal infections are negatively regulated by Cbl-b ubiquitination.