Taming a wild beast: Developing molecular tools and new methods to understand the biology of Zymoseptoria tritici.

Septoria blotch of wheat is one of the world's most serious plant diseases, which is difficult to control due to the absence of durable host resistance and the increasing frequency of fungicide-resistance. The ascomycete fungus that causes the disease, Zymoseptoria tritici, has been very challenging to study. This special issue of Fungal Genetics and Biology showcases an integrated approach to method development and the innovation of new molecular tools to study the biology of Z. tritici. When considered together, these new methods will have a rapid and dramatic effect on our ability to combat this significant disease.

Introduction

This special issue of Fungal Genetics and Biology describes a comprehensive effort to develop methods and expertise to tackle one of the world’s most serious fungal diseases of wheat. The series of papers within this issue are designed to provide a resource for a new generation of plant pathologists who we hope will be inspired to investigate the biology of Septoria blotch of wheat and develop new and durable disease control strategies.

Wheat is the world’s most widely cultivated crop and is responsible for providing about a quarter of the calories to humankind. Global wheat production was more than 700 million tons in 2013, with the European Union and China producing the largest harvests, and wheat being cultivated across most of the temperate regions of the planet (Gurr and Fones, 2015; Rudd et al., 2015). As well as being the most widely grown crop, wheat is also the most traded food on the international markets – indeed more wheat is traded than all other crops combined. Along with rice and maize, wheat production is therefore critical to global food security. There are many threats to wheat productivity, including the availability of high quality land for cultivation, which is threatened by urbanisation, the sustainable use of fertilisers, which are used in enormous quantities, especially in Europe, and the prevalence of diseases. Wheat diseases, such as Septoria blotch, provide an ever-present threat to wheat production and if they could be controlled effectively, would provide a much-needed boost to productivity that will be required to satisfy increasing global demand for food in the next two decades (Courbot et al., 2015; Gurr and Fones, 2015; Hane et al., 2015; Torriani et al., 2015).

What then are the essential pre-requisites to allow us rapidly to understand the biology of Zymoseptoria tritici? What requirements are necessary to allow a fungal pathogen to be regarded as a model system (Perez-Nadales et al., 2014) and for new insights into the underlying mechanisms of infection and virulence to be understood? The last 20 years have seen pathogens, such as the corn smut fungus Ustilago maydis and the rice blast fungus Magnaporthe oryzae, for example, emerge as model systems (Dean et al., 2012). In both cases, this required the development of many tools, which took many years. It is by considering these issues that this collection of reviews and primary publications has been put together.

Taming a beast – where to begin?

Arguably, the first pre-requisite to being able to investigate any disease is a comprehensive description of the life cycle of the organism and the spatial and temporal dynamics of the infection process. For Z. tritici this means a cell biological investigation of what happens from the moment a spore germinates on the leaf surface, to its location of a stoma and invasion of underlying leaf tissue. Using live cell imaging approaches, facilitated by the tools generated and described elsewhere in this special issue, the cell biology of infection is addressed, with an emphasis on the developmental biology of the fungus, its dimorphic growth habit, its ability to perceive and respond to the leaf surface, and its ability to undertake developmental transitions during its establishment of a wheat infection (Steinberg, 2015). Without a ‘road-map’ of how an infection proceeds, it is difficult to identify points of disease intervention or to understand how any single gene product, or family of proteins (from the pathogen or its host), might be important in disease.

The next essential pre-requisite for the model pathogen tool-kit is the ability to test the function of any gene. Without an ability to construct mutants, it is hard to carry out any reverse genetic approach to test the importance of any selected biological process. Therefore, a high frequency transformation system is essential, with a ready supply of selectable marker genes to allow multiple constructs to be expressed, and the ability to carry out targeted mutations (Kilaru and Steinberg, in 2015; Kilaru et al., 2015a; Schuster et al., 2015a,b; Mehrabi et al., 2015). The latter can be dramatically improved by development of a strain of Z. tritici in which the non-homologous DNA end-joining pathway is impaired (Sidhu et al., 2015a,b). This provides the means to generate targeted gene replacement mutants at very high frequency. Coupled to this, it would be advantageous to have a means by which the function of a gene could be attenuated but without complete loss of function, such as virus-induced gene silencing, and the ability to regulate genes driven by a set of highly controllable promoters by which gene expression can be predictably controlled (Kilaru et al., 2015c; Lebrun et al., 2015; Lee et al., 2015). The latter will also provide the means to over-express, or mis-time the expression of a given gene to tests its function, or requirement (Cairns et al., 2015).

Next, a means by which gene products can be localised using expression of fluorescently-labelled fusion proteins is essential, preferably with optimised fluorescent markers, calibrated and tested for the pathogen and in amenable vectors, with easy cloning strategies for ready construction (Kilaru et al., 2015c; Mehrabi et al., 2015; Schuster et al., 2015a; Sidhu et al., 2015a,b). This provides the means to carry out live cell imaging of a pathogen undergoing infection and directly observing the position, fate and turnover of a gene product, which is absolutely pivotal to understanding its function. But this will make no sense without some context, so having the ability to unequivocally identify organelles, such as nuclei, ER, Golgi bodies, peroxisomes, and mitochondria is essential (Schuster et al., 2015b; Kilaru et al., 2015b; Guo et al., 2015a,b). Also, to understand intracellular trafficking it is necessary to be able to identify and track the movement of endosomes, secretory vesicles and the associated components of the actin and microtubule cytoskeleton and their corresponding motor proteins, so that the transport of proteins and the regulation of such processes can be studied (Guo et al., 2015a,b; Kilaru et al., 2015c; Schuster et al., 2015b). When considered together, these tools will therefore allow rapid elucidation of gene function in Z. tritici, as rapidly as in any fungal pathogen studied today.

Taming the pathosystem

So far, of course, we have only considered addressing the functions of individual genes as opposed to gene families and the function of the whole genome and the corresponding, context-dependent proteomes that are expressed during pathogenesis. To address these requires first of all a detailed understanding of the genome (Goodwin et al., 2011; Testa et al., 2015) and its inherent, strain-to-strain variation, and how this correlated with pathotype. Understanding the role, for example, of accessory, or supernumerary chromosomes, as opposed to the core, invariant gene repertoire of the genome will be key (McDonald et al., 2015). An ability to analyse global patterns of gene expression, proteomic and metabolomics data sets during infection, will also be necessary so that the total expressed proteome can be defined and sub-sets associated with the effector repertoire of the pathogen, proteins required for symptom development and those necessary for fungal proliferation can be readily classified, define and then functionally analysed (Ben M’Barek et al., 2015a,b). Methods to define transcriptional networks by chromatin immuno-precipitation coupled to next generation sequencing to define targets genes downstream of transcription factors, will also allow networks of gene expression to be distinguished readily (Soyer et al., 2015). The ability to then define protein–protein interactions between fungal proteins, but also between fungal proteins that act upon host plant proteins, is also essential, with the ready availability of yeast two-hybrid libraries and associated bespoke protocols to Z. tritici (Ma et al., 2015)

Finally, of course we need to consider the plant host and within this context, transgenic wheat lines expressing organelle-specific markers are already available and being constructed to augment host-pathogen cell biological analysis, while detailed analysis of sources of resistance can guide plant breeding strategies such that durable combinations of resistance genes can be identified and introduced into elite, high yielding commercial wheat cultivars (Brown et al., 2015). With wider knowledge of the fungal pathogen population and prevailing virulence specificities, should some the ability to breed for exclusion of the most prevalent, prevailing pathotypes of the fungus (Vallet et al., 2015), providing a direct route to disease control at least in the medium term.

From tools to understanding

The new methods and resources presented in this special issue mean that the Z. tritici research community now has, arguably, the same level of research tools as any fungal model system. This is remarkable, because it has happened in a very short period of time in response to an acute need, articulated by growers and the agricultural biotechnology industry. An integrated programme has been funded and undertaken to carry out this method development and to begin to apply the newly acquired expertise to understanding this disease. Many research questions immediately present themselves. What is the purpose of the morphogenetic plasticity exhibited by Z. tritici and how are these dimorphic transitions regulated? What is their role in virulence? How does the fungus locate stomata and how is its subsequent growth regulated? What conditions the latent phase of infection by Z. tritici? How is the balance between biotrophic proliferation and disease symptom expression determined? Which plant functions are targeted by Z. tritici during plant infection? How is long-distance control of pathogenesis-associated gene expression achieved and how does the fungus perceive and respond to the internal plant tissue environment during infection? How is sporulation triggered and correctly regulated? All of these questions can now be addressed and the answers provided will result not only in many of the fungicide targets of the future, but also perhaps to some longer-term strategies for broad spectrum disease control. So, to those of you reading this commentary who have never previously considered studying Z. tritici, or perhaps not even considered studying a fungal pathogen before, think of the obstacles that have just been removed. It could be a lot of fun.