Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game.

Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.

A two genes - for - one gene interaction between Leptosphaeria maculans and Brassica napus.

Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene-for-gene' complementarities and how they diversified to increased complexity in the course of plant-pathogen co-evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome-assisted map-based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat-rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein-protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins.

Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway.

SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named 'phytolongins' (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the 'Phyl domain'. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway.

The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus.