Exposing an extensive armoury of effectors
Xia Yan and Bozeng Tang from Nick Talbot's research group at The Sainsbury Laboratory recently published two papers that offer new insights into the effector proteins used by the rice blast fungus to overcome host immune defences. The most comprehensive study of its effector repertoire was provided to date, after which a novel regulator of effector gene expression was identified - linking the pre-penetration stage of the blast fungus with events after host cell invasion.
Group leader, Nick Talbot, says, “We are excited about being able to define the repertoire of Magnaporthe effectors in much greater detail and use this information to identify how effector genes are regulated so precisely during plant infection."
"It is clear that expressing effector-encoding genes at exactly the right time is critical to their function and that transcriptional regulation of effector genes is therefore pivotal to successful plant infection. We hope in future to be able to define the transcriptional machinery that orchestrates the entire battery of Magnaporthe effectors.”
Rice infected with blast disease. Credit: Nick Talbot
Why study Magnaporthe?
Maize, wheat, and rice are the main grass crops that humans rely on for food, providing about half of the world's caloric intake. Unfortunately, our dependence on these crops has also led to the spread of some of the most destructive fungal plant pathogens. One such pathogen is Magnaporthe oryzae which causes rice blast disease and poses a serious threat to global food security.
Magnaporthe oryzae infects rice plants in three main steps: spores land on the plants, specialized cells called appressoria penetrate host tissues, and then the pathogen thrives, spreads, and eventually kills the host to feed before sporulating to spread further. During this process, the fungus secretes effector proteins that facilitate infection.
Schematic representation of each stage of rice blast disease development when genes (in color-coded corresponding Weighted correlation network analysis modules) are co-expressed. Source: Yan et al. 2023
Exposing Magnaporthe's armoury
The process of rice blast infection is of great interest to Nick Talbot’s research group. So much so that, recently, two papers were published by the group in quick succession to provide more insight into the effector proteins the rice blast fungus releases into the plant cell to overcome host immune defences.
The first paper, led by Xia Yan, carried out the most comprehensive study of these rice blast effectors to date.
Xia and colleagues from the Talbot group set out to identify the major changes in gene expression that occur during rice blast disease development and used this information to define the repertoire of fungal effector proteins that are deployed by the fungus during plant infection.
Across a six-day infection time course, ten modules of co-expressed fungal genes were identified. This frequently corresponded to key phases in the pathogen's life cycle, capturing well-characterized genes involved in specific physiological processes as well as broader metabolic shifts and provided evidence of a sequential delivery of secreted proteins during plant infection.
After this, the researchers aimed to expose the complete effector repertoire of M. oryzae, which includes all the predicted secreted proteins expressed during the infection.
By using a higher-resolution RNA-seq method, the authors identified an impressive 863 differentially accumulated transcripts which encode predicted secreted proteins, with 546 being annotated as Magnaporthe effector proteins (MEP).
Xia and co-authors developed a novel fitness assay that can be used to define the contribution of an individual effector to pathogenesis based on 32 representative MEP genes.
By building effector fusions with the Green Fluorescent Protein gene (GFP) they were able to assess subcellular localization and stage-specificity and validate the secretion of several effectors in planta. It was revealed that Mep effectors primarily utilize a common unconventional secretory pathway and are directed towards the cytoplasm of rice cells through the biotrophic interfacial complex.
The rice plasma membrane is invaginated and accumulates at the biotrophic interfacial complex (BIC) during plant infection. The purple fluorescence signal from secreted Mep1-mCherry is surrounded by the green fluorescence signal of the rice cell plant plasma membrane marker LTi6B-GFP as the fungus invades new cells, but the initial epidermal cell is occupied and then loses viability, and Mep1-mCherry fluorescence fills the rice cell. Arrows indicate the BICs in the invaded neighboring cells. Source: Yan et al. 2023.
Of particular interest, a novel effector Mep1 was discovered as conferring a fitness advantage to M. oryzae during rice colonization. This highlights the importance of this research in identifying and functionally cataloguing fungal effectors.
This study reveals a diverse repertoire of effectors which is essential for a successful infection, as well as the significant changes in gene expression associated with blast disease.
Xia says, “We’re very excited for future discoveries that can be made with this large-scale dataset, which provides a comprehensive view of the M. oryzae-rice interaction and the effector repertoire involved.”
What exactly happens after Magnaporthe lands on a leaf?
We know that these effector-encoding genes are only expressed when the blast fungus is infecting the plant, and not during other stages of its development.
This has made scientists curious about how the M. oryzae can regulate its effector genes so precisely after it senses its host plant.
With the impressive dataset of blast effectors from Yan et al. in hand, Bozeng Tang and researchers from the Talbot group and the University of Exeter were able to explore the regulation of effectors by developing a very simple forward-genetic screen.
A method was employed to identify regulatory genes responsible for effector expression in M. oryzae during plant infection.
This involved generating a battery of M. oryzae mutant strains expressing the effector-encoding genes fused to the Green Fluorescent Protein gene (GFP) since they are specifically expressed by M. oryzae during infection. Mep2 is one of them.
Having established that Mep2-GFP is mainly expressed in the invasive hyphae of the fungus, UV mutagenesis was carried out on conidia of the Mep2-GFP strain and mutants with constitutive fluorescence in conidia were selected. This was based on the assumption that the mutation induces, or de-represses, expression of the MEP2 effector gene in that location.
Bulked segregant analysis and genetic complementation defined rgs1cer7 as the allele responsible for constitutive expression of Mep2-GFP in conidia. Source: Tang et al. 2023
To identify the specific mutation leading to constitutive MEP2 expression, and therefore the locus of the regulator gene, the researchers performed bulked segregation. Only one mutation was found, and Bozeng and his team were surprised to find that it was in the gene for the regulator Rgs1.
Rgs1 is well known as a regulator of appressorium in M. oryzae, but how it regulates effector expression is unclear.
Further analyses revealed that a large group of effectors are suppressed by the same regulator and are temporally co-expressed at the late stage during infection. This is reciprocal to the regulator Rgs1, which is expressed in conidia but diminished during infection. Thus, Rgs1 not only regulates appressoria formation before penetration, but also supresses effectors during invasive growth.
Live-cell imaging was performed during a time course experiment to investigate expression of Rgs1-GFP in conidia, germ tubes, mature appressoria, and invasive hyphae. Credit: Tang et al. 2023.
Targeted deletion and over-expression of Rgs1 affected the pathogenicity and long-term fitness of M. oryzae, respectively. This shows that Rgs1 is a master regulator required for the correct temporal dynamics of effector gene expression which plays a crucial role in the successful invasion of the plant by the fungus, tying in both developmental biology and morphogenetics.
Bozeng says, “This work highlights how sophisticated the strategy employed by the pathogen is for facilitating successful colonization in the plant. Our findings also share a glimpse of the patterns at play during this critical phase of rice blast infection."
He adds, "We are very excited to provide the research community with a simple experimental design of forward genetics to find effector regulation mechanisms, allowing for more discovery in this relatively underexplored field. Camilla Molinari, a PhD student in the Talbot group, will pursue this line of investigating by exploring more effector regulators through screening.”
Fundamental discoveries in the minutia of plant-microbe interactions can lead to very impactful real-world solutions. The identification of a master regulator during the infection process, such as Rgs1, provides a potential drug target for controlling rice blast disease, with major implications for global food security.
Funding
Both projects were supported by the European Research Council and The Gatsby Charitable Foundation. Bozeng Tang was supported by a Halpin Scholarship and Xia Yan's study was supported by the BBSRC Institute Strategic Programme Grant in Plant Health BBS/E/J/000PR9797 and grant BB/V016342/1.
Research summaries were initially drafted with the help of ChatGPT.
