Peter A. Anderson

Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Plant residues — A low cost, effective bioremediation treatment for petrogenic hydrocarbon-contaminated soil

Petrogenic hydrocarbons represent the most commonly reported environmental contaminant in industrialised countries. In terms of remediating petrogenic contaminated hydrocarbons, finding sustainable non-invasive technologies represents an important goal. In this study, the effect of 4 types of plant residues on the bioremediation of aliphatic hydrocarbons was investigated in a 90 day greenhouse experiment. The results showed that contaminated soil amended with different plant residues led to statistically significant increases in the utilisation rate of Total Petroleum Hydrocarbon (TPH) relative to control values. The maximum TPH reduction (up to 83% or 6800 mg kg(-1)) occurred in soil mixed with pea straw, compared to a TPH reduction of 57% (4633 mg kg(-1)) in control soil. A positive correlation (0.75) between TPH reduction rate and the population of hydrocarbon-utilising microorganisms was observed; a weaker correlation (0.68) was seen between TPH degradation and bacterial population, confirming that adding plant materials significantly enhanced both hydrocarbonoclastic and general microbial soil activities. Microbial community analysis using Denaturing Gradient Gel Electrophoresis (DGGE) showed that amending the contaminated soil with plant residues (e.g., pea straw) caused changes in the soil microbial structure, as observed using the Shannon diversity index; the diversity index increased in amended treatments, suggesting that microorganisms present on the dead biomass may become important members of the microbial community. In terms of specific hydrocarbonoclastic activity, the number of alkB gene copies in the soil microbial community increased about 300-fold when plant residues were added to contaminated soil. This study has shown that plant residues stimulate TPH degradation in contaminated soil through stimulation and perhaps addition to the pool of hydrocarbon-utilising microorganisms, resulting in a changed microbial structure and increased alkB gene copy numbers. These results suggest that pea straw in particular represents a low cost, effective treatment to enhance the remediation of aliphatic hydrocarbons in contaminated soils.

Engineering Saccharomyces cerevisiae To Release 3-Mercaptohexan-1-ol during Fermentation through Overexpression of an S. cerevisiae Gene, STR3, for Improvement of Wine Aroma

ABSTRACT Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5′-phosphate-dependent cystathionine β-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Significance Fungal and oomycete pathogens cause devastating diseases in crop plants and facilitate infection by delivering effector molecules into the plant cell. The secreted effector protein AvrM from flax rust, a fungal pathogen that infects flax plants, internalizes into host cells in the absence of the pathogen, binds to phosphoinositides, and is recognized directly by the resistance protein M in flax to initiate effector-triggered immunity. We describe the crystal structure of AvrM and identify functionally important surface regions in the protein, which advances our understanding of the molecular mechanisms underlying how effectors enter host cells and how they are detected by the plant immune system. Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

Significance Plants and animals use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to defend themselves against invading microbes. In this study, we report the solution structure of the N-terminal coiled-coil (CC) domain from the wheat stem rust resistance protein Sr33. Remarkably, this structure differs substantially from the published crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein MLA10. Using a structural, biophysical, and functional approach, we compare the Sr33 CC domain with other structurally defined NLR CC domains. Collectively, this work redefines our current understanding of the structure and function of plant NLR CC domains, which has significant implications for future studies into this important class of defense receptors. Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model

Correlation between cell death signaling, nucleotide binding, and ligand binding activities of plant immune receptor variants suggests activation is based on altering the equilibrium between ON/OFF states. NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.

Multiplex PCR-based reverse line blot assay for simultaneous detection of 22 virulence genes in uropathogenic Escherichia coli

ABSTRACT Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.

Asexual genetic exchange in the barley pathogen Rhynchosporium secalis

ABSTRACT The causal agent of barley scald, Rhynchosporium secalis, is a haploid anamorphic ascomycete with no known sexual stage. Nevertheless, a high degree of genetic variation has been observed in fungal populations on commercial barley cultivars and parasexuality has been suggested to contribute to this variation. In order to test whether asexual genetic exchange can occur, isolates of R. secalis were transformed to hygromycin B resistance or phleomycin resistance. Mixtures of transformants were co-inoculated either on agar or in planta and screened for the occurrence of dual-antibiotic-resistant colonies. No dual-antibiotic-resistant colonies resulted from mixing transformants of different fungal isolates. In contrast, with transformants originating from the same fungal isolate, asexual exchange of markers was demonstrated on agar plates and in planta. This is the first definitive evidence of asexual genetic exchange in R. secalis.

Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the enzymatic removal of haze-forming grape pathogenesis-related proteins using added proteases. The major problem with this approach is that grape pathogenesis-related proteins are highly protease resistant unless they are heat denatured in combination with enzymatic treatment. This paper demonstrates that the protease BcAP8, from the grape fungal pathogen Botrytis cinerea , is capable of degrading chitinase, a major class of haze-forming proteins, without heat denaturation. Because BcAP8 effectively removes haze-forming proteins under normal winemaking conditions, it could potentially benefit winemakers by reducing bentonite requirements.