Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

ABSTRACTIn filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility.IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Role of Calcium Signaling Genes in Heterokaryon Incompatibility in Neurospora crassa

We have studied the Ca2+-signaling knockout mutants for their role in mating-type-associated heterokaryon incompatibility in Neurospora crassa. The found results showed on heterokaryons homokaryosis for DNCU05225, DNCU06366, DNCU06650, DNCU07075, and ∆NCU07966 Ca2+-signaling knockout mutants (Neurospora crassa unit number, NCU) displayed heterokaryon het compatibility; however heterokaryons heterokaryosis for DNCU05225, DNCU063665, DNCU06650, DNCU07075, and ∆NCU07966 mutants displayed het incompatibility like the wild-type control. In addition to that Two Ca2+-signaling knockout mutants DNCU02283, and DNCU09655 were tested for mating-type-associated heterokaryon incompatibility; these results showed, heterokaryons homokaryosis and heterokaryons heterokaryosis for DNCU02283, DNCU09655 mutants displayed het incompatibility. Cell death and hyphal compartmentation due to mating type associated incompatibility were confirmed by uptake of vital dye Evan’s blue. Thus, these results of NCU05225, NCU06366, NCU06650, NCU07075, and NCU07966 Ca2+-signaling gene products could play a role in mating-type-associated heterokaryon incompatibility in N. crassa. In this article, we are reporting initially screened Ca2+-signaling gene deletion mutants of these five acts as recessive suppressors of mating type associated vegetative incompatibility in N. crassa.Int J Appl Sci Biotechnol, Vol 3(4): 668-679

Programmed Cell Death in Neurospora crassa

Programmed cell death has been studied for decades in mammalian cells, but simpler organisms, including prokaryotes, plants, and fungi, also undergo regulated forms of cell death. We highlight the usefulness of the filamentous fungus Neurospora crassa as a model organism for the study of programmed cell death. In N. crassa, cell death can be triggered genetically due to hyphal fusion between individuals with different allelic specificities at het loci, in a process called “heterokaryon incompatibility.” Chemical induction of cell death can also be achieved upon exposure to death-inducing agents like staurosporine, phytosphingosine, or hydrogen peroxide. A summary of the recent advances made by our and other groups on the discovery of the mechanisms and mediators underlying the process of cell death in N. crassa is presented.