Ammonia leakage can underpin nitrogen-sharing among soil microorganisms.

Soil microbial communities host a large number of microbial species that support important ecological functions such as biogeochemical cycling and plant nutrition. The extent and stability of these functions are affected by inter-species interactions among soil microorganisms, yet the different mechanisms underpinning microbial interactions in the soil are not fully understood. Here, we study the extent of nutrient-based interactions among two model, plant-supporting soil microorganisms, the fungi Serendipita indica, and the bacteria Bacillus subtilis. We found that S. indica is unable to grow with nitrate - a common nitrogen source in the soil - but this inability could be rescued, and growth restored in the presence of B. subtilis. We demonstrate that this effect is due to B. subtilis utilising nitrate and releasing ammonia, which can be used by S. indica. We refer to this type of mechanism as ammonia mediated nitrogen sharing (N-sharing). Using a mathematical model, we demonstrated that the pH dependent equilibrium between ammonia (NH3) and ammonium (NH+4) results in an inherent cellular leakiness, and that reduced amonnium uptake or assimilation rates could result in higher levels of leaked ammonia. In line with this model, a mutant B. subtilis - devoid of ammonia uptake - showed higher S. indica growth support in nitrate media. These findings highlight that ammonia based N-sharing can be a previously under-appreciated mechanism underpinning interaction among soil microorganisms and could be influenced by microbial or abiotic alteration of pH in microenvironments.

Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.

Molecular cloning of ATR5(Emoy2) from Hyaloperonospora arabidopsidis, an avirulence determinant that triggers RPP5-mediated defense in Arabidopsis.

RPP5 is the seminal example of a cytoplasmic NB-LRR receptor-like protein that confers downy mildew resistance in Arabidopsis thaliana. In this study, we describe the cloning and molecular characterization of the gene encoding ATR5(Emoy2), an avirulence protein from the downy mildew pathogen Hyaloperonospora arabidopsidis isolate Emoy2. ATR5(Emoy2) triggers defense response in host lines expressing the functional RPP5 allele from Landsberg erecta (Ler-0). ATR5(Emoy2) is embedded in a cluster with two additional ATR5-like (ATR5L) genes, most likely resulting from gene duplications. ATR5L proteins do not trigger RPP5-mediated resistance and the copy number of ATR5L genes varies among H. arabidopsidis isolates. ATR5(Emoy2) and ATR5L proteins contain a signal peptide, canonical EER motif, and an RGD motif. However, they lack the canonical translocation motif RXLR, which characterizes most oomycete effectors identified so far. The signal peptide and the N-terminal regions including the EER motif of ATR5(Emoy2) are not required to trigger an RPP5-dependent immune response. Bioinformatics screen of H. arabidopsidis Emoy2 genome revealed the presence of 173 open reading frames that potentially encode for secreted proteins similar to ATR5(Emoy2), in which they share some motifs such as EER but there is no canonical RXLR motif.

Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome.

Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

Hyaloperonospora Arabidopsidis as a pathogen model.

Hyaloperonospora arabidopsidis, a downy mildew pathogen of the model plant Arabidopsis, has been very useful in the understanding of the relationship between oomycetes and their host plants. This naturally coevolving pathosystem contains an amazing level of genetic diversity in host resistance and pathogen avirulence proteins. Oomycete effectors identified to date contain a targeting motif, RXLR, enabling effector entry into the host cell. The availability of the H. arabidopsidis genome sequence has enabled bioinformatic analyses to identify at least 130 RXLR effectors, potentially used to quell the host's defense mechanism and manipulate other host cellular processes. Currently, these effectors are being used to reveal their targets in the host cell. Eventually this will result in an understanding of the mechanisms used by a pathogen to sustain a biotrophic relationship with a plant.