How to Kill an Insect, Fungus Style!

Scientists hunt to uncover new genes and mechanisms involved in insect fungal pathogenesis

Crunch! The familiar sound made as an insect is accidentally crushed beneath your shoe. For us, killing an insect can be that simple, a raising and lowering of our foot; however, for other organisms, like fungi who use insects as food and a means to finish their lifecycle, this approach will not work. So instead, fungi have evolved a more sophisticated method to kill insects, and scientists are really interested in understanding how they do it.

But why do scientists care? What is so important about understanding the way fungal pathogens (or fungi that cause infections) infect insects? Turns out that an understanding of how fungi infect insects can have important applications. For example, one group of parasitic fungi, the Metarhizium species, are being genetically engineered to possibly replace chemical insecticides, or work alongside them, against agricultural pests and disease carrying insects like ticks. Using fungal species as an alternative to chemical pesticides offers an option that will be more environmentally friendly, as long as it is done carefully. That is why scientists strive to identify the best way to genetically edit the fungi to make them unable to persist in the environment or out compete natural populations, while simultaneously making them more infectious towards specific insects.

In a general sense, how fungi infect insects is understood; they grow through them. A fungal pathogen does this by first breaking through the hard exoskeleton, or cuticle, on the outside of an insect’s body. This is achieved when the fungus secretes cuticle-degrading enzymes, which are proteins that help break down the cuticle. In addition to these proteins, the fungus grows a specialized cell that is common in many different fungal pathogens, called the appressorium. This cell acts like a fungal crow bar in that it is used to form mechanical pressure to break through the cuticle. Once through, fungal cells grow as long-branching filaments called hyphae, to assist in penetrating the body cavity, which is where the insect’s circulatory fluids and organs reside. Fungal cells in the body cavity then continue to grow as smaller, rounder cells, while producing fungal toxins to eventually kill the host.

However, in order to maximize the efficiency of these fungi as an insecticide and remove environmental concerns, a much deeper understanding of fungal pathogenesis must be reached at the genetic and molecular level. That was the goal of a group of scientists at Zhejiang University, who in the last year have uncovered a new regulatory process that drives insect fungal pathogenesis.

…in order to maximize the efficiency of these fungi as a pesticide, a much deeper understanding of fungal pathogenesis must be reached at the genetic and molecular level…

In their study, the scientists used Metarhizium robertsii, a frequently studied fungal pathogen, and Galleria mellonella larvae (or greater wax moth) as an insect host to model fungal infections. The scientists focused on trying to understand the regulation of fungal genes during a key point in the infection process – the transition between the cuticle and body cavity – which are two drastically different environments that impose unique stress that must be overcome for the fungus to succeed.

The scientists’ starting point was based on the previous finding of a gene (a sequence of DNA that encodes a protein) called COH1, which is highly expressed (or turned on and producing the encoded protein) when fungal cells are colonizing the body cavity. In this more recent study, the researchers used a molecular method to measure if COH1 is also expressed during cuticle penetration. They found it is not expressed, which means COH1 plays a role in pathogenesis in the body cavity but not the cuticle.

So if COH1 is regulating body cavity colonization, what gene is regulating cuticle penetration? To address this question, the scientists explored if there were any proteins that interacted with the COH1 protein. Using interaction experiments, they discovered a candidate protein which they named COH2. By additional biochemical tests, they confirmed that a COH1-COH2 interaction occurs and that it leads to the breakdown of the COH2 protein. This means COH1 negatively impacts COH2 function.

Well what does COH2 do? The COH2 gene encodes a transcription factor, which is a protein that can regulate the expression of other genes. In this study they explored what genes COH2 regulates and found that it turns on genes that produce cuticle-degrading enzymes and turns off genes related to growing in the body cavity. So it seemed they found a gene regulating cuticle penetration.

But what is preventing COH1 from being turned on when fungal cells are in the cuticle? To test this question, the scientists genetically removed multiple known regulators of gene expression from the fungus’s DNA and found that the expression of COH1 went up when specific ones were removed (like HAT1). This means that the function of HAT1 is to prevent COH1 from turning on.

So overall, the scientists discovered critical insights into the regulation of a fungal pathogen’s transition from the insect cuticle to the body cavity (see model above). They showed that as fungal cells are tunneling through the cuticle, COH2 is promoting cuticle break down and preventing unnecessary genes involved in body cavity colonization from turning on. During this stage, COH1 is kept ‘off’ by HAT1. Once the fungus gets through the cuticle, the gene COH1 is turned on and triggers the breakdown of COH2, thereby shutting off the expression of cuticle-degrading enzymes (which if secreted in the body cavity would cause a host immune response) and allowing genes necessary for growth in the body cavity to be turned on.

The knowledge gained by this study can aid scientists in understanding which mechanisms should be targeted when genetically editing fungal pathogens to behave as insecticides. Another benefit is that insights from this study may help scientists better understand fungal pathogenicity that occurs in other organisms as well (e.g. plants and humans) due to many regulatory mechanisms in fungi being evolutionarily conserved.

…Overall, the scientists discovered critical insights into the regulation of a fungal pathogen’s transition from the cuticle to the body cavity of an insect…

Although this study made major breakthroughs, there are still important questions that remain. Such as, how does the fungus sense when it is in the cuticle versus in the body cavity? What gene(s) are involved in the sensing? Once sensed, how is information about the organism’s location transmitted to ensure COH1 is turned on at the appropriate time? For now, scientists will just have to keep working to uncover these fine details. Identifying the next key gene or mechanism involved in fungal pathogenesis may only be an experiment away.