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.

Iron (Fe) is a ubiquitous redox-active element essential for most life. The formation of localized cell wall appositions, the oxidative burst and the production of pathogenesis-related proteins are hallmarks of plant defense responses. Here, we report that iron is a central mediator linking these three phenomena. We show that in response to pathogen attack, reactive Fe3+, but not Fe2+, is deposited at cell wall appositions where it accumulates and mediates the oxidative burst. We provide evidence that the bulk secretion of Fe3+ provoked by pathogen attack leads to intracellular iron depletion, and that H2O2 itself induces ATP-dependent intracellular iron efflux. Finally, we demonstrate that this intracellular iron depletion promotes the transcription of pathogenesis-related genes in concert with H2O2. This work identifies iron as an underlying factor associated with the oxidative burst and regulating cereal defenses, and establishes links between disease-related iron homeostasis in plants and animals.

The agriculturally important genus Colletotrichum is an emerging model pathogen for studying defense in Arabidopsis. During the process of screening for novel pathogenic Colletotrichum isolates on Arabidopsis, we found significant differences in defense responses between detached and attached leaf assays. A near-adapted isolate Colletotrichum linicola A1 could launch a typical infection only on detached, but not attached, Arabidopsis leaves. Remarkably, resistance gene-like locus RCH1-mediated resistance in intact plants also was compromised in detached leaves during the attacks with the virulent reference isolate C. higginsianum. The differences in symptom development between the detached leaf and intact plant assays were further confirmed on defense-defective mutants following inoculation with C. higginsianum, where the greatest inconsistency occurred on ethylene-insensitive mutants. In intact Arabidopsis plants, both the salicylic acid- and ethylene-dependent pathways were required for resistance to C. higginsianum and were associated with induced expression of pathogenesis-related genes PR1 and PDF1.2. In contrast, disease symptom development in detached leaves appeared to be uncoupled from these defense pathways and more closely associated with senescence: an observation substantiated by coordinated gene expression analysis and disease symptom development, and chemically and genetically mimicking senescence.

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.

To acquire iron from plant hosts, fungal pathogens have evolved at least two pathways for iron uptake. One system is hinged on the secretion and subsequent uptake of low-molecular-weight iron chelators termed siderophores, while the other uses cell-surface reductases to solubilize ferric iron by reducing it to ferrous iron for uptake. We identified five iron uptake-related genes from the head blight pathogen Fusarium graminearum and showed that they were transcribed in response to iron limitation. To examine the relative contribution of the reductive and siderophore pathways of iron uptake, we created mutants disrupted at the ferroxidase gene FET3 (Deltafet3) or the siderophore biosynthetic gene SID1 (Deltasid1). The Deltafet3 mutants produced wild-type amounts of siderophores and grew at the same rate as the wild-type under iron limitation, but accumulated high levels of free intracellular iron. The Deltasid1 mutants did not produce siderophores and grew slowly under low iron conditions. Transcription of the iron uptake-related genes was induced in the Deltasid1 mutant regardless of the growth medium iron content, whereas these genes were transcribed normally in the Deltafet3 mutant. Finally, the Deltasid1 mutants could infect single, inoculated spikelets, but were unable to spread from spikelet-to-spikelet through the rachises of wheat spikes, while the Deltafet3 mutants behaved as wild-type throughout infection. Together, our data suggest that siderophore-mediated iron uptake is the major pathway of cellular iron uptake and is required for full virulence in F. graminearum.

The biotrophic powdery mildew fungus Blumeria graminis releases extracellular materials to the surface of fungal infection structures that facilitate anchoring them to hydrophobic plant surfaces prior to infection; however, the chemistry of fungal adhesives and the mechanism of adhesion remain largely unclear. Expressed sequence tag analysis led to identification of a secreted lipase, Lip1, from B. graminis. Expression of LIP1 is dramatically upregulated during the early stages of fungal development. Lip1, secreted to the surface of fungal cell walls, possesses lipolytic activity against a broad range of glycerides and releases alkanes and primary fatty alcohols from the epicuticular wax of wheat leaves. Of the epicuticular wax components released by Lip1 activity, long-chain alkanes are the most efficient cues for triggering appressorium formation. Pretreatment of wheat leaves with Lip1, thereby removing leaf surface wax, severely compromises components of fungal pathogenicity, including conidial adhesion, appressorium formation, and secondary hypha growth. Our data suggest that Lip1 activity releases cues from the host surface to promote pathogen development and infection.

SUMMARYFindings from multi-year, multi-site field trial experiments measuring maize yield response to inoculation with the phosphorus-solubilizing fungus, Penicillium bilaiae Chalabuda are presented. The main objective was to evaluate representative data on crop response to the inoculant across a broad set of different soil, agronomic management and climate conditions. A statistical analysis of crop yield response and its variability was conducted to guide further implementation of a stratified trial and sampling plan. Field trials, analysed in the present study, were conducted across the major maize producing agricultural cropland of the United States (2005–11) comprising 92 small (with sampling replication) and 369 large (without replication) trials. The multi-plot design enabled both a determination of how sampling area affects the estimation of maize yield and yield variance and an estimation of the ability of inoculation with P. bilaiae to increase maize yield. Inoculation increased maize yield in 66 of the 92 small and 295 of the 369 large field trials (within the small plots, yield increased significantly at the 95% confidence level, by 0·17 ± 0·044 t/ha or 1·8%, while in the larger plots, yield increases were higher and less variable (i.e., 0·33 ± 0·026 t/ha or 3·5%). There was considerable inter-annual variability in maize yield response attributed to inoculation compared to the un-inoculated control, with yield increases varying from 0·7 ± 0·75 up to 3·7 ± 0·73%. No significant correlation between yield response and soil acidity (i.e., pH) was detected, and it appears that pH reduction (through organic acid or proton efflux) was unlikely to be the primary pathway for better phosphorus availability measured as increased yield. Seed treatment and granular or dribble band formulations of the inoculant were found to be equally effective. Inoculation was most effective at increasing maize yield in fields that had low or very low soil phosphorus status for both small and large plots. At higher levels of soil phosphorus, yield in the large plots increased more with inoculation than in the small plots, which could be explained by phosphorus fertilization histories for the different field locations, as well as transient (e.g., rainfall) and topographic effects.

A genetic linkage map of Brassica rapa L. was constructed using recombinant inbred lines (RILs) derived from a cross between yellow-seeded cultivar Sampad and a yellowish brown seeded inbred line 3-0026.027. The RILs were evaluated for seed color under three conditions: field plot, greenhouse, and controlled growth chambers. Variation for seed color in the RILs ranged from yellow, like yellow sarson, to dark brown/black even though neither parent had shown brown/black colored seeds. One major QTL (SCA9-2) and one minor QTL (SCA9-1) on linkage group (LG) A9 and two minor QTL (SCA3-1, SCA5-1) on LG A3 and LG A5, respectively, were detected. These collectively explained about 67% of the total phenotypic variance. SCA9-2 mapped in the middle of LG A9, explained about 55% phenotypic variance, and consistently expressed in all environments. The second QTL on LG A9 was ~70 cM away from SCA9-2, suggesting that independent assortment of these QTLs is possible. A digenic epistatic interaction was found between the two main effect QTL on LG A9; and the epistasis × environment interaction was nonsignificant, suggesting stability of the interaction across the environments. The QTL effect on LG A9 was validated using simple sequence repeat (SSR) markers from the two QTL regions of this LG on a B(1)S(1) population (F(1) backcrossed to Sampad followed by self-pollination) segregating for brown and yellow seed color, and on their self-pollinated progenies (B(1)S(2)). The SSR markers from the QTL region SCA9-2 showed a stronger linkage association with seed color as compared with the marker from SCA9-1. This suggests that the QTL SCA9-2 is the major determinant of seed color in the A genome of B. rapa.