Invasion of the stigma by oomycete pathogenic hyphae or pollen tubes : striking similarities and differences.

Both filamentous pathogens' hyphae and pollen tube penetrate the host's outer layer and involve growth within the host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla of Arabidospis thaliana's stigma, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcomed pollen tube. We showed that Phytophtora parasitica, a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cells, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganisation of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion.

Transcriptome dynamics of Arabidopsis thaliana root penetration by the oomycete pathogen Phytophthora parasitica.

BACKGROUND: Oomycetes are a group of filamentous microorganisms that includes both animal and plant pathogens and causes major agricultural losses. Phytophthora species can infect most crops and plants from natural ecosystems. Despite their tremendous economic and ecologic importance, few effective methods exist for limiting the damage caused by these species. New solutions are required, and their development will require improvements in our understanding of the molecular events governing infection by these pathogens. In this study, we characterized the genetic program activated during penetration of the plant by the soil-borne pathogen Phytophthora parasitica. RESULTS: Using all the P. parasitica sequences available in public databases, we generated a custom oligo-array and performed a transcriptomic analysis of the early events of Arabidopsis thaliana infection. We characterized biological stages, ranging from the appressorium-mediated penetration of the pathogen into the roots to the occurrence of first dead cells in the plant. We identified a series of sequences that were transiently modulated during host penetration. Surprisingly, we observed an overall down regulation of genes encoding proteins involved in lipid and sugar metabolism, and an upregulation of functions controlling the transport of amino acids. We also showed that different groups of genes were expressed by P. parasitica during host penetration and the subsequent necrotrophic phase. Differential expression patterns were particularly marked for cell wall-degrading enzymes and other proteins involved in pathogenicity, including RXLR effectors. By transforming P. parasitica with a transcriptional fusion with GFP, we showed that an RXLR-ecoding gene was expressed in the appressorium and infectious hyphae during infection of the first plant cell. CONCLUSION: We have characterized the genetic program activated during the initial invasion of plant cells by P. parasitica. We showed that a specific set of proteins, including effectors, was mobilized for penetration and to facilitate infection. Our detection of the expression of an RXLR encoding gene by the appressorium and infection hyphae highlights a role of this structure in the manipulation of the host cells.

The Phytophthora parasitica RXLR effector penetration-specific effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology.

Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica. Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes. Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner. We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.

Distinct dynamics of HISTONE3 variants between the two fertilization products in plants.

Sexual reproduction involves epigenetic reprogramming comprising DNA methylation and histone modifications. In addition, dynamics of HISTONE3 (H3) variant H3.3 upon fertilization are conserved in animals, suggesting an essential role. In contrast to H3, H3.3 marks actively transcribed regions of the genome and can be deposited in a replication-independent manner. Although H3 variants are conserved in plants, their dynamics during fertilization have remained unexplored. We overcame technical limitations to live imaging of the fertilization process in Arabidopsis thaliana and studied dynamics of the male-gamete-specific H3.3 and the centromeric Histone Three Related 12 (HTR12). The double-fertilization process in plants produces the zygote and the embryo-nourishing endosperm. We show that the zygote is characterized by replication-independent removal of paternal H3.3 and homogeneous incorporation of parental chromatin complements. In the endosperm, the paternal H3.3 is passively diluted by replication while the paternal chromatin remains segregated apart from the maternal chromatin (gonomery). Hence epigenetic regulations distinguish the two products of fertilization in plants. H3.3-replication-independent dynamics and gonomery also mark the first zygotic divisions in animal species. We thus propose the convergent selection of parental epigenetic imbalance involving H3 variants in sexually reproducing organisms.

The immediate activation of defense responses in Arabidopsis roots is not sufficient to prevent Phytophthora parasitica infection.

SUMMARY: *The outcome of plant-microbe interactions is determined by a fine-tuned molecular interplay between the two partners. Little is currently known about the molecular dialogue between plant roots and filamentous pathogens. We describe here a new pathosystem for the analysis of molecular mechanisms occurring during the establishment of a compatible interaction between Arabidopsis thaliana roots and a root-infecting oomycete. *We performed cytological and genetic analyses of root infection during the compatible interaction between A. thaliana and Phytophthora parasitica. *Phytophthora parasitica uses appressoria to penetrate A. thaliana roots. Initial biotrophic growth is accompanied by the formation of haustoria, and is followed by a necrotrophic lifestyle. Arabidopsis thaliana mutants with impaired salicylic acid (SA), jasmonic acid (JA) or ethylene (ET) signaling pathways are more susceptible than the wild-type to infection. The salicylate- and jasmonate-dependent signaling pathways are concertedly activated when P. parasitica penetrates the roots, but are downregulated during invasive growth, when ethylene-mediated signaling predominates. *Thus, defense responses in A. thaliana roots are triggered immediately on contact with P. parasitica. Our findings suggest that the pattern of early defense mechanism activation differs between roots and leaves.

Strategies of attack and defense in plant-oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan).

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant-pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.

Transcriptome dynamic of Arabidopsis roots infected with Phytophthora parasitica identifies VQ29, a gene induced during the penetration and involved in the restriction of infection.

Little is known about the responses of plant roots to filamentous pathogens, particularly to oomycetes. To assess the molecular dialog established between the host and the pathogen during early stages of infection, we investigated the overall changes in gene expression in A. thaliana roots challenged with P. parasitica. We analyzed various infection stages, from penetration and establishment of the interaction to the switch from biotrophy to necrotrophy. We identified 3390 genes for which expression was modulated during the infection. The A. thaliana transcriptome displays a dynamic response to P. parasitica infection, from penetration onwards. Some genes were specifically coregulated during penetration and biotrophic growth of the pathogen. Many of these genes have functions relating to primary metabolism, plant growth, and defense responses. In addition, many genes encoding VQ motif-containing proteins were found to be upregulated in plant roots, early in infection. Inactivation of VQ29 gene significantly increased susceptibility to P. parasitica during the late stages of infection. This finding suggests that the gene contributes to restricting oomycete development within plant tissues. Furthermore, the vq29 mutant phenotype was not associated with an impairment of plant defenses involving SA-, JA-, and ET-dependent signaling pathways, camalexin biosynthesis, or PTI signaling. Collectively, the data presented here thus show that infection triggers a specific genetic program in roots, beginning as soon as the pathogen penetrates the first cells.

Analysis of molecular markers genetically linked to the Leptosphaeria maculans avirulence gene AvrLm1 in field populations indicates a highly conserved event leading to virulence on Rlm1 genotypes.

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5' end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race "AvrLm1" at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.

A dialogue between the SIRENE pathway in synergids and the fertilization independent seed pathway in the central cell controls male gamete release during double fertilization in Arabidopsis.

Angiosperms sexual reproduction involves interactions between the two female gametes in the embryo sac and the two male gametes released by the pollen tube. The two synergids of the embryo sac express the FERONIA/SIRENE receptor-like kinase, which controls the discharge of the two sperm cells from the pollen tube. FER/SRN may respond to a ligand from the pollen tube. Alternatively, the interaction between FER/SRN and a ligand from the embryo sac may lead to a state of competence of the synergids allowing pollen tube discharge. Here, we report the new mutant scylla (syl) impaired in the control of pollen tube discharge. This mutant also produces autonomous endosperm development in absence of fertilization-a trait associated with the FERTILIZATION INDEPENDENT SEED (FIS) mutant class. This led us to identify autonomous endosperm in srn mutants and to demonstrate synergistic interactions between srn and the fis mutants. In addition, the fis mutants display defects in pollen tube discharge as in srn and syl mutants, confirming the interaction between the two pathways. Our findings suggest that pollen tube discharge is controlled by an interaction between the synergids expressing SRN/FER and the central cell expressing FIS genes.

Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene.

In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.

The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum.

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, Clpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.