Background

Although artificial inoculation of perennial ryegrass (Lolium perenne) is broadly used to study its interaction with Epichloë festucae, there exists no comprehensive protocol for the evaluation of responses to fungal infection at an early post-inoculation time point, which likely defines the future of this interaction. In our recent work (Rahnama et al., 2018), we adapted methods to study four of the most important host plant responses during plant-fungal interactions including cell death, callose deposition, lignin production, and hydrogen peroxide (H₂O₂) production.

In response to invading microbes, one of the early plant actions is the production of different types of reactive oxygen species (ROS), including H₂O₂, which act as an antimicrobial agent to protect the plant against invading microbes and are one of the first signals to induce other plant responses (Walters, 2003). In addition, by depositing callose and lignin, plants remodel their cell wall at the infection site to stop invading fungi (Luna Diez et al., 2010; Zipfel, 2009). As a late defense response, plant cells surrounding the infection site undergo programmed cell death (hypersensitive response) to limit available food for the invading microbe (Lo Presti et al., 2015). To initiate and maintain a successful symbiotic lifestyle, Epichloë endophytes must somehow suppress or avoid these different host defense responses; although, very little is known about how this occurs. Developing methods to detect these responses will aid in the understanding of this system, especially when applied to the study of mutant strains that compromise symbiosis.

To stain for callose deposition, we adapted an Aniline Blue staining method (Knox, 1979) that was originally used for the detection of callose in pollen tubes but has also been used in different fungal-plant interactions such as powdery mildew infection of Arabidopsis (Ellinger et al., 2013). Besides aniline blue, Toluidine Blue O has also been used in other systems such as guava root infection with Fusarium (Gupta et al., 2012). In addition to staining methods, there exist non-staining methods using immunofluorescence that detect callose at the micro-level in internal cell sites such as plasmodesmata (Pendle and Benitez-Alfonso, 2015).

To detect lignin deposition, we used a Safranin solution adapted from a study on maize infection by Ustilago maydis (Tanaka et al., 2014); however, Safranin is broadly used in other systems such as Fusarium infection of different cereals (Knight et al., 2011). Moreover, the Wiesner (phloroglucinol-HCl) reaction (Pomar et al., 2002) is another method to study lignin deposition in plant-fungal interactions.

To visualize cell death responses, Trypan Blue staining is the most widely used method, for which we adapted the protocol used to study Arabidopsis infection with downy mildew (Koch and Slusarenko, 1990). Recently, a non-toxic method has also been suggested, which uses red light imaging (Landeo Villanueva et al., 2021).

There are several methods to detect H₂O₂, one of the basic methods for which is the detection of the oxidization of small molecules, which we also used here. This method is based on visualizing the color change of 3,3’-diaminobenzidine (DAB) after being oxidized by H₂O₂. We adapted this method from a study on barley infection with powdery mildew (Thordal-Christensen et al., 1997). Other recent methods to study H₂O₂ have used genetically encoded green fluorescent protein (GFP)-based probes that react directly with H₂O₂ (reviewed in Winterbourn, 2018).

Here, we demonstrate that these methods are useful for defining the response of perennial ryegrass to infection with Epichloë endophyte at a very early time point. Using these methods in time-point studies, we defined the most suitable time to measure these responses post-inoculation. As such, these methods are useful for studying compatible and incompatible interactions of symbiotic fungi with their host plants during the early stages of infection (Rahnama et al., 2018 and 2019). Although these methods are routinely used for other fungal-plant interactions, our adaption of them to study the early inoculation stages of Epichloë-grass interactions is novel. These methods can be used in other grass-endophyte interactions that use a similar inoculation technique.