Research highlights a novel role for reactive oxygen species in the fungus-grass symbiotic relationship -- finding could help engineer resistance to crop pathogens

Wilde type (left) and noxA mutant (right) perennial reygrass infected with E. festucae fungus.

A symbiotic relationship is one in which two organisms of different species interact in ways that profoundly affect their livelihoods and reproductive success. Such interactions range from mutually beneficial to antagonistic and are considered to be of major ecological and evolutionary importance in shaping plant and animal communities. Examples of beneficial symbioses include the microbes that live in the guts of herbivorous mammals like cows and help to digest cellulose, ants that protect plants from herbivores, and the fig wasps that pollinate fig trees by depositing their eggs in the fig flowers, which their larvae then feed on. Plants participate in numerous symbiotic associations. Examples include the nitrogen-fixing bacteria that live in plant roots, the fungus-alga association that makes up lichens, and grasses and endophytic fungi (fungi that live inside the leaves, stems, and other structures of the plant).

Fungal endophytes in the genus Epichloë form symbiotic associations with many grasses. Studies have shown that Epichloë endophytes can result in enhanced biomass production, seed production, and root growth of the grass plants as well as improved recovery after drought compared to plants without endophytes. Like other endophytes, the symbioses of grass species with Epichloë fungi can be mutualistic or antagonistic or both. In the beneficial interactions, Epichloë endophytes are strictly limited in their intercellular growth throughout the plant. The growth of the endophyte is synchronized with that of the grass; fungal hyphae grow actively in expanding leaves but cease to grow as the leaf matures.

Aiko Tanaka, Daigo Takemotot and Barry Scott at the Centre for Functional Genomics at Massey University in New Zealand; Michael Christensen at the Grasslands Research Centre, also in New Zealand, and Pyoyun Park at the Graduate School of Science and Technology at Kobe University, Japan, studied the interaction of the fungal endophyte Epichloë festucae and its host, perennial ryegrass, Lolium perenne. As a result, they discovered a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between E. festucae and its grass host.

Tanaka et al. used a forward genetics approach to create mutants of the endophyte that would be unable to establish or maintain a mutualistic relationship with perennial ryegrass. They inserted foreign DNA randomly into the genome of Epichloë festucae, resulting in a population of fungal strains having disruptions in different genes throughout the fungal genome. From this collection they isolated a mutant that is unable to synchronize its growth with that of the plant host.

Plants infected with the mutant fungus showed stunted growth, premature senescence, and death, whereas those infected with the wild-type fungus exhibited their usual growth pattern. This was accompanied by a dramatic increase in fungal endophyte growth within the plant compared with plants inoculated with wild-type fungus. The fungal hyphae of the wild type fungus showed limited branching and were mostly oriented parallel to the intercellular spaces of the leaf. On the other hand, the hyphae of the mutant fungus showed extensive colonization of the leaf--similar to a pathogenic infection. As a result, the biomass of the mutant fungus increased significantly compared to wild type. Thus a mutualistic interaction became an antagonistic one with the mutation of a single gene.

Tanaka et al. then went on identify and sequence the fungal gene responsible for the mutant phenotype. They determined that the foreign DNA had disrupted a fungal gene, called noxA, which encodes an enzyme that catalyzes the conversion of molecular oxygen to superoxide. The altered symbiotic phenotype is due to a mutation (caused by the insertion of a segment of foreign DNA) in the E. festucae noxA gene.

NADPH oxidase catalyzes the production of ROS or superoxides by transferring electrons from NADPH (a ubiquitous electron donor in nature) to molecular oxygen, with secondary generation of hydrogen peroxide. Superoxides are unstable and highly reactive molecules that can be extremely destructive in biological systems and have been implicated, for example, as causal agents in cancer formation. For this reason, antioxidants, which destroy superoxides are recommended as cancer prevention measures. However, ROS can be part of the arsenal that plants use to protect themselves, as NADPH oxidase enzymes generate superoxides in response to pathogen colonization.

Tanaka et al. looked at the production of the ROS hydrogen peroxide (H2O2) in plants infected with wild type and mutant E. festucae by electron microscopy. Cerium perhydroxides, which are formed by a reaction with H2O2, were detected in actively growing tissue of plants with wild type fungus but rarely in the same tissue of plants with mutant fungus. These results confirmed that it is the fungus, not the plant, that is mainly responsible for ROS production.

The authors proposed that ROS produced by the endophyte NoxA enzyme in the plant negatively regulates the growth of the fungus, preventing excessive colonization of the host. Thus, the ROS act as a brake on the growth of the fungus, preventing it from becoming pathogenic and allowing it to maintain a beneficial, mutualistic symbiosis with the plant. When this gene is disrupted, the growth of the fungus is uncontrolled and the association becomes pathogenic. This study has highlighted a previously unknown role for ROS in maintaining a mutualistic symbiosis between endophytic fungi and plants and shown that the mutation of the fungal noxA gene can switch the symbiosis from beneficial to antagonistic.

The authors of this study are Aiko Tanaka, Daigo Takemoto, and Barry Scott of the Centre for Functional Genomics, Institute of Molecular BioSciences, Massey University, New Zealand; Michael J. Christensen, AgResearch, Grasslands Research Centre, Palmerston North, New Zealand; and Pyoyun Park, Graduate School of Science and Technology, Kobe University, Japan.

The research paper cited in this report is available at the following link: http://www.aspb.org/pressreleases/TPC039263.pdf

The Plant Cell (http://www.plantcell.org/) is published by the American Society of Plant Biologists. For more information about ASPB, please visit http://www.aspb.org/.