Sweet talk: A plant protein releases a fungal β-glucan to enhance colonization

When plants or animals interact with fungi, the first site of physical contact is often the fungal extracellular polysaccharide (EPS) matrix, a layer that surrounds the cell wall. The EPS matrix is mainly composed of sugars and proteins and it is not surprising that some EPS components modulate immune responses in organisms the fungus interacts with. The functions of β-glucans have been well characterized in animal pathogenic fungi: some act as microbe-associated molecular patterns (MAMPs) that trigger host immune responses, while others have antioxidative properties and do not elicit immunity. In plant-associated fungi, little is known about the composition and function of the EPS matrix; however, it has been established that soluble glycans released from this matrix are likely a general molecular feature of the interaction with plants (Wawra et al., 2019; Wanke et al., 2021).

In a new publication, Balakumaran Chandrasekar, Alan Wanke, Stephan Wawra, and colleagues (Chandrasekar et al., 2022) present a yet undescribed strategy used by both fungal endophytes and parasites to mitigate plant immune defenses through the selective release of a β-glucan with reactive oxygen species (ROS) scavenging activity. The authors developed a fluorescent probe to visualize the presence of 1,3-glucans in the EPS matrix of the beneficial root endophyte Serendipita indica and the pathogenic fungus Bipolaris sorokiniana. Then, they performed a glycosyl-linkage analysis and found that terminal glucose, 3-glucose, and 3,6-glucose were abundant glycosidic linkages in the EPS layer, in contrast to the cell wall, where 4-linked glucose was dominant. How do these linkages affect the interaction with the plant? In a previous publication (Wawra et al., 2016), the same lab had identified a protein in barley (Hordeum vulgare), HvBGLUII, a β-glucanase that is present during fungal colonization. Now, the authors set out to investigate the activity of HvBGLUII on the fungal EPS matrix and cell wall. First, they used thin layer chromatography and found a product that HvBGLUII released from the EPS layer but not from the cell wall. By combining mass spectrometry, glycosyl linkage analysis, and NMR, they were able to identify the product as a β-glucan decasaccharide (β-GD) with seven β-1,3-linked glucose and three β-1,6-glucose units (see Figure). The authors elegantly complemented these findings by treating β-GD with two different enzymes that specifically act on β-1,3 and β-1,6 bonds and hydrolyzed β-GD. Chandrasekar and co-workers then turned to the question of what exactly β-GD does at the fungus–plant interface. They performed ROS burst assays and found that application of β-GD led to a decrease in ROS levels, even in the presence of different MAMPs. Furthermore, the addition of the same two enzymes that were used to digest β-GD restored the ROS burst. They then used a 3,3′-diaminobenzidine (DAB) precipitation assay and mass spectrometry to show that β-GD is a ROS scavenger. Strikingly, when they tested whether HvBGLUII cleaved laminariheptaose, a linear β-1,3-glucan without the β-1,6-linked branches, the enzyme digested it into glucose and laminaribiose, leading to higher ROS accumulation, demonstrating that HvBGLUII acts as a host-defense enzyme but is hijacked and then releases a ROS scavenger during fungal accommodation. Finally, the authors obtained similar results using the distantly related plant pathogen B. sorokiniana, suggesting that the antioxidative properties of β-GD are conserved among plant-associated fungi.

Figure.

HvBGLUII is hijacked to produce β-GD. HvBGLUII usually digests linear β-1,3-glucan, triggering defensive ROS accumulation. In the presence of plant-colonizing fungi, HvBGLUII releases a ROS scavenging β-1,3:1,6-glucan decasaccharide (β-GD) from the fungal EPS matrix, mitigating plant defense and enhancing fungal colonization. Adapted from Chandrasekar et al. (2022), Figure 6.

The results provided by Chandrasekar et al. are a major advancement in the technically challenging field of plant glycan-triggered immunity. Some details remain to be addressed: A β-GD molecule might only be used to scavenge ROS a single time, and in that case, it would be interesting to know how fast the β-GD pool gets depleted. In addition, it would be exciting to see whether β-GD has similar immune properties in plants other than Arabidopsis and barley. Finally, the plant receptors for β-glucans have not been clearly identified yet, and a more comprehensive picture of the complexity of β-glucan structures will certainly facilitate their discovery.