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- Materials and Methods
- Supporting Information
Filamentous plant pathogens – comprising true fungi and oomycetes – have evolved various strategies for entering their hosts (Mendgen et al., 1996). Some enter via wounds or natural openings, such as stomata. Others penetrate their host by enzymatic or mechanical means, using specialized structures called appressoria. Most of the information about appressorium differentiation and function has been obtained from true fungi, such as the ascomycete Magnaporthe grisea. Differentiation of the M. grisea appressorium is induced by the perception of surface hardness and hydrophobicity, which activates the cyclic AMP (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways (Lee et al., 2003; Caracuel-Rios & Talbot, 2007). Autophagic cell death is then required to mobilize lipids and carbohydrates from the spore and to generate the mechanical force of the appressorium (Veneault-Fourrey & Talbot, 2007). However, the function of the appressorium has not yet been fully characterized and whole-genome microarrays are currently being used to investigate overall changes in gene expression (Oh et al., 2008).
Little is known about host penetration by oomycetes. They were long considered to be fungi, because of their filamentous growth. Consequently, the data obtained for ascomycetes were generally extrapolated to oomycetes. Phytopathogenic oomycetes include more than 85 Phytophthora species, hundreds of Pythium species and downy mildews. They infect many different host plants, including crops, ornamental plants and natural populations (Erwin & Ribeiro, 1996). Only a few studies have investigated the penetration process of oomycetes. It was shown that poor nutrient content, surface hydrophobicity and topography induce appressorium differentiation in Phytophthora palmivora (Bircher & Hohl, 1997). However, appressorium differentiation in Phytophthora, unlike that in true fungi, requires calcium but is not induced by cAMP (Bircher & Hohl, 1999). This difference suggests that different signaling pathways are activated in fungi and oomycetes. Differential screening and proteomic studies in Phytophthora infestans and Phytophthora sojaehave shown that a specific genetic program is activated during appressorium differentiation (Krämer et al., 1997; Avrova et al., 2003; Ebstrup et al., 2005; Chen et al., 2007). Genes involved in protein synthesis and amino acid and energy metabolism are induced during appressorium development and early infection (Ebstrup et al., 2005; Grenville-Briggs et al., 2005). A gene expression profiling study performed on P. infestans demonstrated the accumulation of transcripts encoding protein kinases, cell wall-degrading enzymes (CWDEs) and various effectors – proteins used to manipulate plant cells – during appressorium differentiation (Judelson et al., 2008). Only three Phytophthora gene functions have been shown to be specifically required for the penetration process. Silencing of the Pibzp1 transcription factor from P. infestans abolishes appressorium differentiation (Blanco & Judelson, 2005). The RNAi-mediated silencing of a family of four cellulose synthase genes from P. infestans has also been shown to impair appressorium differentiation and plant infection (Grenville-Briggs et al., 2008). The PIHMP1 gene encodes a membrane protein that accumulates in appressoria and haustoria and is required for early infection (Avrova et al., 2008).
The oomycete penetration process has been analyzed on artificial substrates that cannot be pierced (Bircher & Hohl, 1997). Thus, functions occurring during plant cell wall breaching and the initial exchanges with the host may not have been detected. Moreover, such studies were performed using pathogens infecting aerial parts of plants while most pathogenic oomycetes infect plants via the roots. The few studies of root infection have generated conflicting results. P. sojaeinfects soybean (Glycine max) via hyphae rather than appressoria (Enkerli et al., 1997). Conversely, an apical swelling at the penetration site has been observed in P. parasitica/citrus, Phytophthora palmivora/citrus, Phytophthora megasperma/Cicer arietinum and Phytophthora cryptogea/Chrysanthemum morifolium interactions (Swiecki & Donald, 1988; Dale & Irwin, 1991; Enkerli et al., 1997; Widmer et al., 1998). Root penetration by an oomycete has yet to be fully characterized.
A new inoculation assay was used to characterize the P. parasitica penetration process. This species infects the roots of a wide range of plants and is thus representative of most pathogenic oomycetes. A cytological analysis of early infection showed that P. parasitica entered host cells using an appressorium. We generated a cDNA library for this oomycete at the penetration stage and showed the accumulation of sequences encoding functions associated with pathogenicity. We then monitored the accumulation of transcripts encoding CWDE and ‘RXLR-EER’ effectors.
- Top of page
- Materials and Methods
- Supporting Information
We describe the process by which P. parasitica penetrates its host. We show that the initial invasion of the plant root is mediated by an appressorium. This structure, which differentiates at the tip of the germ tube, develops as a round cell that both redirects growth and breaches the plant cell wall. This is consistent with previous cytological analyses describing hyphal swellings at the penetration sites of soil-borne Phytophthora species (Swiecki & Donald, 1988; Dale & Irwin, 1991; Enkerli et al., 1997) and shows that P. parasitica invades its hosts by differentiating a specialized structure. Like Kramer and coworkers, we observed a septum separating the appressorium from the germ tube, showing that this structure is definitively different from a hyphae (Krämer et al., 1997).
As in most filamentous root pathogens, analyses of the P. parasitica penetration process are limited by the asynchronous development of infection structures and low pathogen biomass at early stages. By means of a penetration assay based on onion epidermis, we synchronized the early events of infection and obtained appressoria highly similar to those observed on tomato roots.
Using chloroform-treated dead onion epidermis to induce appressorium differentiation, we obtained a cDNA library from P. parasitica at the penetration stage with a higher proportion of pathogen sequences (79%) than that reported for other plant–Phytophthora EST collections, even from later stages of infection during which the oomycete biomass is much greater (Qutob et al., 2000; Randall et al., 2005).
Putative pathogenicity-related sequences appeared to be relatively abundant (7% of the single-sequence set). The transcripts of genes encoding proteins potentially involved in protection against ROS or in the export of plant toxic compounds were overrepresented with respect to the P. parasitica or P. sojae libraries generated from mycelia isolated from the plant or grown in vitro (Torto-Alalibo et al., 2007; Le Berre et al., 2008). Already described for P. infestans or M. grisea appressoria differentiated in vitro, the expression of such functions during early infection would enable the pathogen to deal with the plant’s early defense responses (Ebstrup et al., 2005; Judelson et al., 2008; Oh et al., 2008).
We identified 36 CWDE-encoding sequences, most of which were absent from the published P. parasitica mycelium and late infection-derived EST libraries (Le Berre et al., 2008). Quantitative RT-PCR experiments confirmed that the transcripts of 21 of these genes accumulated preferentially early in infection (zoospore to appressorium stages), with preferential accumulation in the appressoria observed for nine of these sequences. Similar results were obtained for some P. infestans CWDE (Judelson et al., 2008). Thus, expression of a specific set of CWDEs in the P. parasitica appressorium may be required to soften the plant cell wall or may be involved in pathogen cell wall remodeling during the penetration process, as suggested for an M. grisea cutinase mutant impaired in penetration peg formation (Skamnioti & Gurr, 2007). The P. parasitica appressorium library contains sequences encoding cellulose synthases. As cellulose synthesis is required for P. infestans host penetration (Grenville-Briggs et al., 2008), it would be of interest to determine the mechanisms governing the balance between cellulose degradation and synthesis in the early stages of infection with Phytophthora.
We also tried to identify effectors potentially exported to the plant cell wall or cytoplasm to favor pathogen development. The proportion of secreted proteins (5.6%) was similar to that deduced from the P. infestans, P. sojae and P. ramorum proteomes (4.5, 7.8 and 7.5%, respectively). Apoplastic effectors, such as elicitin-like proteins, CRN-like proteins and Nep1-like proteins, were as abundant in our cDNA library as in libraries for the necrotrophic stages of P. parasitica and P. sojae, indicating that these genes may be expressed very early in infection (Kamoun, 2006; Torto-Alalibo et al., 2007; Le Berre et al., 2008). We also identified at least six sequences encoding proteins containing the RXLR-EER motif found in potential effectors and oomycete avirulence genes, and identified seven other sequences that may also belong to this family. Transcripts corresponding to three of these effectors preferentially accumulated in the appressoria, as already observed for similar proteins from P. infestans (Whisson et al., 2007; Judelson et al., 2008). At least two RXLR proteins are exported from the haustoria into the plant cytoplasm and abolish plant cell death (Bos et al., 2006; Whisson et al., 2007; Dou et al., 2008). The secretion of such proteins from appressoria remains to be confirmed, but may be involved in the abolition of plant defense responses or the modification of plant cells to facilitate invasion by the pathogen.
According to our quantitative RT-PCR experiments, most of the transcripts accumulating in P. parasitica appressoria differentiated on tomato also accumulated in appressoria differentiated on onion epidermis, showing that our inoculation assay is reliable for analyses of the penetration process. The few exceptions observed could account for host-specific responses or for loss of induction of some genetic pathways as a consequence of penetration of dead onion tissues. This limitation will have to be considered in future studies.
Sixty per cent of the appressorium-derived sequences were not present in previous P. parasitica EST collections (Shan et al., 2004; Skalamera et al., 2004; Panabières et al., 2005; Le Berre et al., 2008). Furthermore, our quantitative RT-PCR experiments showed that some transcripts accumulate specifically in the appressoria, and that genes expressed at the penetration stage are not always expressed early in zoospores or in artificial cyst germinations (diluted culture medium) as sometimes proposed (Shan et al., 2004; Torto-Alalibo et al., 2007). We show here that a specific genetic program operates at the appressorium stage.
The onion epidermis penetration assay provides molecular and cytological data concerning appressorium-mediated host penetration by P. parasitica. The synchronization of this process and the strong enrichment in pathogen sequences observed render this assay particularly useful for the identification of transcripts involved in the initial infection of plant cells. Several putative pathogenicity factors were identified and future studies will focus on identification of the genes required for host cell penetration by P. parasitica. Most of the ESTs identified in the P. parasitica appressorium library were also present in P. sojae, which is thought to penetrate host cells without the need for an appressorium (Enkerli et al., 1997). It would therefore be of interest to determine the role of these sequences in host penetration by P. sojae and in P. parasitica, in a comparative study.