Hitchhiker's guide to multi-dimensional plant pathology

Authors


  • Diane Saunders was a finalist for the 2014 New Phytologist Tansley Medal for excellence in plant science, which recognises an outstanding contribution to research in plant science by an individual in the early stages of their career; see the Editorial by Lennon & Dolan, 205: 951–952.

Summary

Filamentous pathogens pose a substantial threat to global food security. One central question in plant pathology is how pathogens cause infection and manage to evade or suppress plant immunity to promote disease. With many technological advances over the past decade, including DNA sequencing technology, an array of new tools has become embedded within the toolbox of next-generation plant pathologists. By employing a multidisciplinary approach plant pathologists can fully leverage these technical advances to answer key questions in plant pathology, aimed at achieving global food security. This review discusses the impact of: cell biology and genetics on progressing our understanding of infection structure formation on the leaf surface; biochemical and molecular analysis to study how pathogens subdue plant immunity and manipulate plant processes through effectors; genomics and DNA sequencing technologies on all areas of plant pathology; and new forms of collaboration on accelerating exploitation of big data. As we embark on the next phase in plant pathology, the integration of systems biology promises to provide a holistic perspective of plant–pathogen interactions from big data and only once we fully appreciate these complexities can we design truly sustainable solutions to preserve our resources.

The event horizon of the plant–pathogen interface

As the world population increases it is imperative that sustainability of crop yields is addressed to meet the needs of an ever-expanding global population. Filamentous pathogens pose a substantial threat to global food security. A severe epidemic affecting all five of the most widely consumed crops (rice, potato, wheat, maize and soybean) simultaneously could leave over half the world's population hungry (Fisher et al., 2012). Therefore, minimizing losses due to pathogens would make significant inroads towards achieving global food security. The first step is to attain a better understanding of the microbial enemies we face.

One central question in plant pathology is how pathogens cause infection and manage to evade or suppress plant immunity to promote disease. This interplay between pathogen invasion and host immune responses is a multilayered process. The first contact lies at the highly regulated process of host-cell entry. The use of molecular cytology to study this intimate interaction has not only revealed that the process of infection is elegantly orchestrated, but also brought to light many essential regulatory components. Once the host is penetrated, the pathogen suppresses plant defenses and manipulates plant physiology using an arsenal of secreted proteins, known as effectors. The biochemical and molecular study of effector proteins and their function in planta has greatly impacted on our mechanistic understanding of the plant–pathogen interface. All of these processes are ultimately encoded within the genome of the pathogens and their hosts. With rapid advances in DNA sequencing technology over the past decade, genomics is now embedded within the toolbox of next-generation plant pathologists. By employing a multidisciplinary approach plant pathologists can fully leverage these technical advances to answer key questions in plant pathology, aimed at achieving global food security.

This review highlights the impact of multidisciplinary approaches for the study of plant–pathogen interactions, including the impact of (i) cell biology and genetics on progressing our understanding of infection structure formation on the leaf surface, (ii) biochemical and molecular analysis to study how pathogens subdue plant immunity and manipulate plant processes through effectors, (iii) genomics and DNA sequencing technologies on all areas of plant pathology and (iv) new forms of collaboration on accelerating exploitation of big data.

Exploring strange new worlds of pathogen invasion

The ability of filamentous plant pathogens to cause disease is dependent on successful entry to their hosts. To comprehend how pathogens colonize plants, the course of infection must be observed not only at the macroscopic level, but also at the cellular level. Current techniques targeting the cellular level include the use of morphological mutants, fluorescent microscopy and chemical inhibitors of cellular development.

In order to facilitate host tissue entry, many filamentous pathogens develop highly specialized infection structures that are common among cereal fungal pathogens such as the causal agents of rust diseases, downy mildews and the rice blast fungus Magnaporthe oryzae. This fungus has been widely studied as a model to understand the mechanisms that regulate the formation and function of these structures (Fig. 1). Once the three-celled M. oryzae asexual conidium lands on the rice leaf surface, it adheres to the cuticle through secretion of spore tip mucilage from the apex of the conidium (Ebbole, 2007). Cell wall hydrophobins such as Mpg1 further contribute to the adhesion of the conidium to the hydrophobic leaf surface (Talbot et al., 1996). The perception of host cues leads to initiation of a single polarized germtube that elongates, hooks and then differentiates at the tip to form a highly specialized, dome-shaped appressorium. Concurrently, a closed mitotic division occurs either within the germ tube or the terminal cell of the conidium (Saunders et al., 2010a). One daughter nucleus then migrates into the incipient appressorium and one migrates back to the original conidial cell (Veneault-Fourrey et al., 2006; Saunders et al., 2010a). Formation of a septum between the appressorium and germ tube completes cellular division, which is essential for pathogenesis (Saunders et al., 2010b). Efflux of glycerol from the appressorium is prevented by deposition of melanin, which forms a dense inner layer between the chitin-rich cell wall and plasma membrane (Chumley & Valent, 1990). Activation of an Mps1 mitogen-activated protein (MAP) kinase cell-wall integrity pathway enables cell wall remodeling to occur (Xu et al., 1998). The re-location of storage products such as lipids to the appressorium is accompanied by nonselective macroautophagic cell death of the conidium (Veneault-Fourrey et al., 2006; He et al., 2012). The axis of polarity is re-orientated and a penetration peg forms at the base of the appressorium, within a wall-less nonmelanised region (Ebbole, 2007). Remodeling of the F-actin cytoskeleton is a pre-requisite to penetration peg emergence and is coordinated by assembly of septin GTPases at the appressorial pore (Dagdas et al., 2012). NADPH oxidases play a key role in inducing assembly of septin GTPases (Ryder et al., 2013). An enormous amount of turgor pressure develops in the melanised appressorium that is translated into mechanical force to rupture the plant epidermis and enable pathogen colonization.

Figure 1.

Known mechanisms regulating the Magnaporthe oryzae infection cycle. (a) The three-celled M. oryzae asexual conidium lands on the rice leaf surface and adheres to the rice cuticle. (b) The perception of host cues leads to initiation of a single polarized germ tube from the terminal cell. (c) The germ tube elongates, hooks and then begins to differentiate at the tip to form a highly specialized, dome-shaped appressorium. (d) Concurrently, a closed mitotic division occurs either within the germ tube or the terminal cell of the conidium. One daughter nucleus then migrates into the incipient appressorium and one migrates back to the original conidial cell. (e) Formation of a septum between the appressorium and germ tube completes cellular division at 6–8 h post-infection (hpi). (f) Efflux of glycerol from the appressorium is prevented by deposition of melanin, which forms a dense inner layer between the chitin-rich cell wall and plasma membrane. (g) The re-location of storage products such as lipids to the appressorium is accompanied by nonselective macroautophagic cell death of the conidium. (h) An enormous amount of turgor pressure develops in the melanised appressorium that is translated into mechanical force to rupture the plant epidermis and enable pathogen colonization. (i) The axis of polarity is re-orientated and a penetration peg forms at the base of the appressorium through the pore wall overlay, within a wall-less nonmelanised region at 20–24 hpi. (j, k) Disease lesions form on the infected leaf surface 72–96 hpi and under humid conditions conidiospores are produced. Conidia are released from these aerial conidiophores and carried to new host plants. Blue boxes, the key internal processes that regulate appressorium development; orange box, external cues that regulate germination; h, hours post-infection. Loosely based on Ebbole (2007).

The multistep process of appressorium development in M. oryzae is not only dependent on external cues such as surface hardness, hydrophobicity, cuticular wax and lack of exogenous nutrients (Ebbole, 2007), but also exquisitely orchestrated through internal regulatory processes such as cyclic AMP signaling, a MAP kinase cascade and progression through the cell cycle (Fig. 1). For instance, addition of high extracellular concentrations of cAMP to germinating conidia on noninductive hydrophilic surfaces can induce appressorium development (Lee & Dean, 1993). Analysis of the CPKA gene that encodes a catalytic subunit of the cAMP-dependent protein kinase A, illustrated a role for cAMP signaling in host penetration as ΔCPKA mutants were reduced in pathogenicity but still formed appressoria (Xu et al., 1997). When combined, this analysis implicates cAMP signaling in both surface recognition and host penetration. Downstream of the cAMP signal a Pmk1 MAP kinase pathway is required to initiate appressorium formation and later for invasive growth in planta (Xu et al., 1997). The numerous components identified in the Pmk1 MAP kinase signaling pathway are reviewed in Li et al. (2012).

Following surface recognition, progression through the cell cycle is required for various morphological transitions (Veneault-Fourrey et al., 2006; Saunders et al., 2010a). Addition of hydroxyurea or the use of the temperature-sensitive mutant MoNIM1 to inhibit DNA replication revealed that this phase is essential for initiating the morphogenic transition from polarized growth of the germ tube to isotropic growth of the appressorium (Veneault-Fourrey et al., 2006; Saunders et al., 2010a). Furthermore, use of the never-in-mitosis and blocked-in-mitosis NimA and MoBIM1 temperature-sensitive mutants illustrated that appressorium differentiation is likely to be controlled at the G2-M border (Veneault-Fourrey et al., 2006; Saunders et al., 2010a). At a semi-restrictive temperature the MoBIM1 mutant displayed a significant reduction in virulence indicating that an additional yet uncharacterized cell cycle checkpoint may mediate morphological transitions downstream of appressorium development, such as penetration peg emergence (Saunders et al., 2010a).

Focused at the cellular level, these studies have greatly enhanced our understanding of the regulatory mechanisms that tightly coordinate appressorium morphogenesis, which underpins the ability for M. oryzae to pursue a pathogenic lifestyle.

Resistance is futile: plant cells must be assimilated

Plant resistance against pathogens consists of two tiers of immunity: (1) pattern recognition immune receptor-triggered immunity that acts as the first layer of defense, and (2) intracellular immune receptor-triggered immunity that acts as a second line of defense (Win et al., 2012). Pathogens overcome these barriers by modulating and subverting plant innate immune responses to enable parasitic infection. To this end, pathogens secrete an array of effectors within host cells (Petre & Kamoun, 2014). However, some effectors are recognized by plant immune receptors in particular host genotypes and resistance responses are initiated against the invading pathogen, turning these effectors into avirulence factors (Petre & Kamoun, 2014). Advancement of our knowledge in this molecular arms race has relied upon traditional biochemical and molecular techniques to search for host targets of these effector proteins, their corresponding immune receptors, and the translocation machinery pivotal to their entry into host cells. These themes form the basis of current research directed towards understanding the mechanisms that underpin this highly complex interaction.

Functional characterization of virulence and avirulence alleles of effectors can provide clues about how pathogens may evade host recognition. For instance, the potato late blight oomycete pathogen Phytophthora infestans AVR2 effector associates and forms a complex with the host protein BSL1. BSL1 can interact with the corresponding immune protein R2 leading to resistance (Saunders et al., 2012a). However, the epidemic clonal P. infestans lineage blue13 expresses a virulent AVR2 variant. This variant still associates with the host target BSL1 but fails to promote the interaction of BSL1 with R2 and evades R2-mediated recognition (Saunders et al., 2012a). Therefore, mutation could act as a powerful evolutionary force, particularly for potential ‘core’ effectors that are always present (typically with polymorphisms) and expressed during infection across genetically diverse pathogen genotypes. This provides a mechanistic view of how effectors with avirulence activity maintain their virulence activity whilst evading recognition by immune receptors in addition to mechanisms such as deletion or pseudogenization. Better knowledge of pathogen effector recognition should prove useful in managing and improving the longevity of corresponding resistance genes.

An example of an effector that manipulates a host physiological process to the advantage of the pathogen is Tin2 from Ustilago maydis. The Tin2 protein was shown to induce anthocyanin biosynthesis by stabilizing and activating the host ZmTTK1 kinase enzyme (Tanaka et al., 2014). This redirects the phenylalanine-derived intermediate metabolites required for lignin biosynthesis towards anthocyanin biosynthesis. A reduction in lignin deposition in vascular bundle cells within the host potentially facilitates access of the pathogen to nutrients (Tanaka et al., 2014). Pursuing host targets of effector proteins provides a better understanding of how effectors may hijack plant processes and evade host recognition.

Effectors must be translocated into host cells to interact with their target proteins. To date, little is known about the process of effector translocation (Petre & Kamoun, 2014). Evidence of pathogen molecules being detected within plant cells was shown by studies of the Uf-RTP1p protein from the bean rust fungus Uromyces fabae (Kemen et al., 2005). Using immunofluorescence and electron microscopy the authors detected Uf-RTP1p at the host–microbe interface and inside infected plant cells to varying degrees dependent on infection stage (Kemen et al., 2005). Studies of effector proteins from P. infestans have indicated the requirement of a conserved motif for host translocation for one class of effectors (Whisson et al., 2007). This motif is characterized by the amino acid sequence RXLR, frequently followed by EER. Replacement of the RXLR-EER motif in the P. infestans avirulence effector protein Avr3a by KMIK-DDK prevented its delivery into plant cells (Whisson et al., 2007). More recently, a controversial mechanistic view of effector translocation has been proposed to involve binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P; Kale et al., 2010). This requires clarification as it is yet to be confirmed universally for other oomycetes (Petre & Kamoun, 2014). In M. oryzae effector translocation requires a highly localized pathogen structure called the biotrophic interfacial complex (BIC; Khang et al., 2010). The authors showed that the M. oryzae effectors PWL2 and BAS1 are secreted into the BIC before translocation into the rice host cytoplasm (Khang et al., 2010). In fungal systems no consensus for requirement of a translocation motif has been identified. Due to the critical nature of the effector translocation mechanism, this topic is hotly debated but difficult to study with current methods.

Charting the galaxy

The genomics era has revolutionized research, with bioinformatics at the heart of many of the most progressive ventures in science. Its arrival has brought high-throughput, sequence-driven approaches to the forefront in designing strategies for studying plant–pathogen interactions. Just as the PCR changed the landscape of molecular biology, DNA sequencing technology is shifting the paradigm. Scientists can now freely explore pathogens at the genomic level, searching for signatures that may convey their ability to cause disease. The accessibility of genome sequences has had an immediate impact on the study of pathogen development by identifying conserved regulatory elements and structural mechanisms. Whole genome sequences are also exploited to systematically predict the full effector complement of a pathogen, providing new insights into how these pathogens manipulate their hosts.

Global-scale dissection of developmental stages of filamentous plant pathogens is now possible at greater resolution due to advances in sequencing technology. For instance, transcriptome profiling during infection provides insights into the mechanisms that regulate disease progression. For Colletotrichum higginsianum, a hemibiotrophic fungal pathogen of Brassicaceae, transcriptomic studies revealed that pathogenicity-related genes were activated in three successive waves during pathogenic transitions (O'Connell et al., 2012). Specifically the upregulation of genes encoding secondary metabolities during appressoria formation identified a previously unknown role for infection structures in delivering an array of small molecules to the first infected cells that likely act in host manipulation (O'Connell et al., 2012). Transcriptional profiling of the obligate biotrophic poplar rust pathogen Melampsora larici-populina revealed differential control of carbohydrate-active enzymes, lipases, transporters and proteases at 48 h post-inoculation (Duplessis et al., 2011). By contrast, small-secreted proteins were expressed dynamically throughout infection (Duplessis et al., 2011). These examples illustrate the impact of genomics and transcriptomics on the developmental studies of these pathogens.

Identifying effectors and deciphering their biochemical activities to understand how pathogens successfully colonize and reproduce on their hosts, has become a principal directive in plant pathology. Every pathogen genome sequence published is accompanied by the identification of a putative effector repertoire. The first step is the prediction of secretory signals in the proteome because almost all known effectors of eukaryotic plant pathogens are secreted through the general secretory pathway via the recognition and cleavage of an N-terminal signal peptide. Next, recurrent features that characterize microbial effectors are used to search the predicted secretome and refine the list of candidate effectors (Saunders et al., 2012b). Once the effector repertoire is predicted, these proteins can be used in high-throughput biological assays for avirulence activity. This includes direct delivery into plant cells by exploiting the bacterial type III delivery system or the use of viral vectors delivered indirectly by agrobacteria. Additionally, predicted effector proteins can be used to identify associated host proteins and the underlying physiological processes that are manipulated by the pathogen. This is achieved by (1) expressing effector proteins in planta and affinity purifying them with their associated host target proteins (Saunders et al., 2012a) or (2) using effector candidates in yeast-two-hybrid assays, with selected plant immune genes or broad libraries of host genes to identify interacting partners. The latter method was used to identify molecular hubs in the plant immune system targeted by effectors from pathogens of two different kingdoms, Pseudomonas syringae a bacterium and Hyaloperonospora arabidopsidis an oomycete (Mukhtar et al., 2011). For plant genomes new technologies such as resistance gene enrichment sequencing (RenSeq; Jupe et al., 2013) and the CRISPR/Cas system (Shan et al., 2013) are accelerating the cloning of new resistance genes and hastening the speed of plant breeding. Genomics has enabled these large-scale studies to be realized, vastly improving our knowledge of the mechanistic mystery that is the plant–pathogen arms race.

One for all and all for one

Clearly the impact of the genomic era is far-reaching and generates an overwhelming amount of data. As we move forward, the integration of open-access approaches will be vital to make sure the abundance of information generated is fully exploited, particularly when tackling emerging pathogen threats.

Despite huge advances in sequencing capacity, researchers are still struggling to exploit big data to its full potential. Research specialism often prevents us appreciating the panoramic view afforded by the generation of genomic and transcriptomic data. This calls for a coordinated community response to ensure we gain as much insight as possible from the availability of an unprecedented wealth of data for key pathogens that presently threaten global food security. With the growth in social media portals that provide virtual communities without geographical constraints, now is the time to increase the development of interdisciplinary collaboration and utilize people power to leverage these big datasets (Maclean et al., 2013). We should take heed from our bacteriologist colleagues who spearheaded the concept of ‘crowdsourcing’ to rapidly accelerate genome annotation of the Escherichia coli O104:H4 strain that killed c. 50 people in Germany in 2011 (Rohde et al., 2011). Crowdsourcing involves the use of collaborative open-source networks, which provides researchers an unprecedented opportunity to rally scientists across disciplines to ensure rapid progress from genomic data acquisition to the annotation needed to address key biological questions. In plant pathology, crowdsourcing was recently implemented to study the emergence of Chalara dieback of Ash in the UK, which poses a very real threat to the UK's 80 million Ash trees, regarded as a national icon (Maclean et al., 2013). With the ability to understand our adversary paramount and the cost and speed of genome sequencing decreasing, generating and publically releasing sequence data is key to accelerating research on plant pathogens by soliciting the vast expertise and knowledge of the crowd in the annotation phase.

To boldly go where no one has gone before

I envisage a future where the collective intelligence of the entire scientific community is embraced and applied to all areas of research. As we embark on the next phase in plant pathology, synthetic biology is rapidly becoming an integrated component of our toolbox (Fig. 2). Synthetic biology combines gene synthesis technology with modern engineering design to create new customizable solutions to counter threats to global food security. The final unifying component in our toolbox is the inter-disciplinary field of systems biology. This promises to provide a holistic perspective of plant–pathogen interactions from big data and only once we fully appreciate these complexities can we design truly sustainable solutions to preserve our resources.

Figure 2.

Some of the key components in the plant pathology toolbox. As research in plant pathology has evolved, many key components in the traditional toolbox have been adapted to take full advantage of emerging technologies. For the 21st Century plant pathologist an array of new tools and means of communication are also at our disposal. By employing a multidisciplinary approach plant pathologists can fully leverage these technical advances to answer key questions in plant pathology. Clip art images used with permission from Microsoft.

Acknowledgements

D.G.O.S. is supported by a fellowship in computational biology at The Genome Analysis Centre, in partnership with the John Innes Centre, and strategically supported by BBSRC.

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