HOST SUSCEPTIBILITY

Traditionally, research in the field of plant pathology has focused on incompatible interactions - deciphering the host components associated with disease resistance and the pathogen factors that trigger host defenses. More recently, increasing attention has been focused on host and pathogen factors that promote compatible interactions and disease development (Chrisholm et al. 2006).

We study the interactions between the model host plant iArabidopsis thaliana and the obligate fungal pathogen Golovinomyces cichoracearum, the causal agent of the powdery mildew disease on Arabidopsis (Fig. 1). Because the powdery mildew pathogens have a biotrophic lifestyle, requiring living plant tissues for survival, we reasoned that the host plant is likely to be an active partner in compatible interactions leading to disease. Ways in which the host might contribute to disease development include the formation of structural components of the haustorial complex, such as the extrahaustorial membrane (Fig. 2). Also, since the powdery mildew pathogen draws water, mineral and organic nutrients from its host via the haustorium, adjustments in host metabolism to accommodate the additional demand by the pathogen are likely to be required.

Fig. 2. Cartoon of powdery mildew-infected epidermal cell. The haustorium is a fungal feeding structure, which is surrounded by an uncharacterized extrahaustorial matrix and is encased in a specialized host membrane, the extrahaustorial membrane. cn, conidium, eh, extrahaustorial, ha, haustorium.

We have taken several approaches to determining host contributions to the powdery mildew disease development. Descriptive experiments to define changes in transcript levels using DNA microarrays and changes in subcellular compartments as monitored in Arabidopsis lines carrying GFP-marked organelles have been conducted. In addition, mutational strategies to identify host susceptibility genes by their ability to limit powdery mildew growth when mutated have been conducted.

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Powdery Mildew Resistant (pmr) Mutants

To identify host susceptibility factors, a direct screen for loss-of-susceptibility mutants was conducted. Such mutants appear powdery mildew resistant and can be readily identified among susceptible (wild-type) individuals 7-9 days post-inoculation with the powdery mildew pathogen. The mutants from the screen (n=26) were placed into six complementation groups (Fig. 3). In each case, the mutant gene was genetically recessive suggesting that the mutation could be "loss-of-function" mutation. All six of the mutant classes have been described (Nishimura et al. 2003, Vogel and Somerville 2000, Vogel et al. 2002, Vogel et al. 2004).

Fig. 3. Powdery mildew disease phenotypes of pmr mutants. From left to right: representative leaves from Col (susceptible wild type), pmr1, pmr2, pmr3, pmr4, pmr5, and pmr6, shown 10 days after inoculation with the Arabidopsis powdery mildew, G. cichoracearum.

In a collaborative project with Ralph Panstruga (Max Planck Institute, Cologne, Germany), we discovered that PMR2 (=MLO2) is the primary functional orthologue of the barley gene MLO, a negative regulator of defenses against the barley powdery mildew disease (Consonni et al. 2006). Two related Arabidopsis genes, MLO6 and MLO12 appear to play a minor supporting role to MLO2. Loss-of-function mutants of the barley MLO gene are powdery mildew resistant and Arabidopsis mlo2 mutants exhibit many of the same features as their barley counterparts. The defenses elicited in mlo mutants act early in the infection sequence primarily to limit penetration by the powdery mildews into epidermal cells. Interestingly, mutations in the PEN1 (syntaxin), PEN2 (glycosyl hydrolase) or PEN3 (ABC transporter) suppress penetration resistance in mlo2 mutants. As noted under the section on Non-Host Resistance, the pen1-pen3 Arabidopsis mutants were recovered in a screen for mutants compromised in resistance to inappropriate pathogens (e.g., Arabidopsis resistance to Blumeria graminis f. sp. hordei, barley powdery mildew). Thus, it would appear that the resistance mechanisms under negative control by MLO contribute to resistance to both inappropriate and appropriate pathogens.

The PMR4 gene was found to encode a wound- and pathogen-associated callose synthase (Nishimura et al. 2003). Callose deposition in cell wall appositions, called papillae, just below penetration sites is a common defense response in plants. Thus, it was unexpected that plants deficient for the wound- and stress-inducible callose synthase would be highly disease resistant. We discovered that the basis for this resistance was a hyper-activation of the salicylic acid defense pathway in infected pmr4 plants. How a deficiency in the stress-inducible callose synthase and callose production de-represses (or activates) the salicylic acid pathway remains to be discovered.

The pmr5 and pmr6 mutants are very similar morphologically. The PMR6 gene encodes a novel pectate lyase-like protein and is predicted to localize to the outer surface of the plasma membrane (Vogel et al. 2002), while the PMR5 gene encodes a novel protein predicted to be in the endoplasmic reticulum (Vogel et al. 2004). Double mutant analysis demonstrated that pmr5- and pmr6-based resistances operate independently of the known defense signal transduction pathways (i.e., the salicylic acid and the ethylene/jasmonate pathways). The predicted function of PMR6 as a cell wall degrading enzyme led to an analysis of the cell walls in pmr5 and pmr6. Fourier transform infrared spectrometry indicated that the pectin composition of cell walls from both mutants was altered, suggesting that these two genes participate in pectin biosynthesis, modification or deposition in some manner. Pectin components play important roles in determining the porosity and ionic environment of the cell wall, both features that could influence the exchange of signaling molecules and nutrients between host and pathogen (Vincken et al. 2003). Furthermore, some pectin polymers contain latent elicitors that can be released by pathogen cell wall degrading enzymes during the penetration step (Vorwerk et al. 2004). Thus, the loss of PMR5 or PMR6 may lead to the activation of a novel defense pathway. Alternatively, the powdery mildew pathogen may not longer properly recognize the pmr5 or pmr6 plants as suitable hosts or the powdery mildew may not be able to efficiently penetrate the epidermal wall due to the change in cell wall. An active area of interest in the lab is to determine which of these hypothesized mechanisms might confer resistance in the pmr5 and pmr6 mutants.

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Powdery Mildew-Induced Lesion (mil) Mutants

Among the mutants recovered along with the powdery mildew resistance pmr mutants was a class of mutants that form necrotic and/or chlorotic patches several days following infection by the powdery mildew pathogen (Fig. 4). These mutants were also resistant or partially disease resistant. Seventeen mutants were retained of which the mil1 and mil4 are the best characterized to date.

A variety of abiotic stresses, such as wounding, heat and cold stress, or drought, failed to elicit necrosis or chlorosis in mil1 or mil4 mutants. Inoculation with the virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 did not elicit lesion formation. Among the various treatments, only inoculation with the inappropriate pathogen, barley powdery mildew, also induced necrosis several days after inoculation. The MIL1 (=EDR2) gene was cloned by T-DNA tagging and found to encode a novel gene with three predicted distinct domains -- a StAR (steroidogenic acute response protein) transfer domain, which is a lipid/sterol-binding domain; a pleckstrin homology domain binds to phosphatidylinositol-4-phosphate; and a plant-specific domain of unknown function (DUF1336) (Tang et al. 2005). The MIL4 gene encodes a novel small protein with four transmembrane domains and two splice variants.

It is notable that many of the classic components of programmed cell death in animal systems cannot be readily identified among plant genes. Thus, it would appear that plants may have evolved independent mechanisms for regulating and executing cell death during development and in response to environmental factors. The MIL genes may help us understand the cell death program triggered during plant-pathogen interactions.

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Subcellular Responses to Powdery Mildew Infection

Cutler et al. (2000) have developed a set of Arabidopsis lines each carrying a different GFP tag for a different organelle or subcellular structure. Lines with GFP tagged endoplasmic reticulum, nucleus, cytoplasm, plasma membrane, Golgi, vacuoles and peroxisomes exist. With these lines, infection related changes in the subcellular components can be monitored in real time in living host tissues providing us with our first glimpses of changes in organelle shape and distribution in response to pathogen infection. Powdery mildew infections are particularly suited to such studies because the infections are confined to the leaf surface and epidermal layer and because the pathogen does not kill infected cells.

Fig. 5. Vacuolar-GFP line 20 h.p.i. with powdery mildew. Red, propidium iodide stained fungus; green, GFP tonoplast marker. C, conidium, H, haustorium. The tonoplast is appressed to the haustorium (arrowhead). Adjacent to the penetration site, the vacuole appears vesiculated (arrow).

A few previous studies with epidermal peels monitored changes in subcellular components in living plant tissue but these observations were limited to those that could be made with Nomarski optics in the absence of staining (see review Aist and Bushnell 1991). In agreement with this literature, GFP-tagged organelles accumulated around the haustorial complex (Koh et al. 2005). This was most dramatic with the peroxisomes, which appear to move preferentially toward haustoria. However, all other subcellular structures appeared to aggregate near the haustorium (Figure 5). In addition, the GFP-plasma membrane marker lines dramatically illustrate that the extrahaustorial membrane, the plant membrane encasing the haustorial complex, is distinct from the plasma membrane (Koh et al. 2005). How the extrahaustorial membrane is formed and what the permeability properties of this membrane are will be important questions to address in future experiments, as the formation of this specialized membrane is key to successful fungal infection.

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