Isabelle Fudal

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans.

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.

Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.

Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen.

Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices.

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, a...

Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins.

The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.

The APSES transcription factor LmStuA is required for sporulation, pathogenic development and effector gene expression in Leptosphaeria maculans

Leptosphaeria maculans causes stem canker of oilseed rape (Brassica napus). The APSES transcription factor StuA is a key developmental regulator of fungi, involved in morphogenesis, conidia production and also more recently described as required for secondary metabolite production and for effector gene expression in phytopathogenic fungi. We investigated the involvement of the orthologue of StuA in L. maculans, LmStuA, in morphogenesis, pathogenicity and effector gene expression. LmStuA is induced during mycelial growth and at 14 days after infection, corresponding to the development of pycnidia on oilseed rape leaves, consistent with the function of StuA described so far. We set up the functional characterization of LmStuA using an RNA interference approach. Silenced LmStuA transformants showed typical phenotypic defects of StuA mutants with altered growth in axenic culture and impaired conidia production and perithecia formation. Silencing of LmStuA abolished the pathogenicity of L. maculans on oilseed rape leaves and also resulted in a drastic decrease in expression of at least three effector genes during in planta infection, suggesting either that LmStuA regulates, directly or indirectly, the expression of several effector genes in L. maculans or that the infection stage in which effectors are expressed is not reached when LmStuA expression is silenced.

Chromatin-based control of effector gene expression in plant-associated fungi

Plant-associated fungi often present in their genome areas enriched in repeat sequences and effector genes, the latter being specifically induced in planta. The location of effector genes in regions enriched in repeats has been shown to have an impact on adaptability of fungi but could also provide for tight control of effector gene expression through chromatin-based regulation. The distribution of two repressive histone marks was shown to be an important regulatory layer in two fungal species with different lifestyles. Chromatin-based control of effector gene expression is likely to provide an evolutionary advantage by preventing the expression of genes not needed during vegetative growth and allow for a massive concerted expression at particular time-points of plant infection.

Effector Biology in Fungal Pathogens of Nonmodel Crop Plants

The recent finding of a novel fungal strategy to manipulate salicylic acid (SA) in a nonmodel plant pathogen interaction not only establishes the universality of the strategy to ensure the success of biotrophs and hemibiotrophs, but also illustrates current limitations and challenges to identify targets of fungal effector in crop plants.