Characterization of Phn15.1, a Newly Identified Phytophthora nicotianae Resistance QTL in Nicotiana tabacum.

Phytophthora nicotianae is an oomycete that causes black shank, one of the most economically important diseases affecting tobacco production worldwide. Identification and introgression of novel genetic variability affecting partial genetic resistance to this pathogen is important because of the increased durability of partial resistance over time as compared with genes conferring immunity. A previous mapping study identified a quantitative trait locus (QTL), hereafter designated as Phn15.1, with a major effect on P. nicotianae resistance in tobacco. In this research, we describe significantly improved resistance of nearly isogenic lines (NILs) of flue-cured tobacco carrying the introgressed Phn15.1 region derived from highly resistant cigar tobacco cultivar Beinhart 1000. The Phn15.1 region appeared to act in an additive or partially dominant manner to positively affect resistance. To more finely resolve the position of the gene or genes underlying the Phn15.1 effect, the QTL was mapped with an increased number of molecular markers (single-nucleotide polymorphisms) identified to reside within the region. Development and evaluation of subNILs containing varying amounts of Beinhart 1000-derived Phn15.1-associated genetic material permitted the localization of the QTL to a genetic interval of approximately 2.7 centimorgans. Importantly, we were able to disassociate the Beinhart 1000 Phn15.1 resistance alleles from a functional NtCPS2 allele(s) which contributes to the accumulation of a diterpene leaf surface exudate considered undesirable for flue-cured and burley tobacco. Information from this research should be of value for marker-assisted introgression of Beinhart 1000-derived partial black shank resistance into flue-cured and burley tobacco breeding programs.

Accelerated flowering time reduces lifetime water use without penalizing reproductive performance in Arabidopsis.

Natural selection driven by water availability has resulted in considerable variation for traits associated with drought tolerance and leaf-level water-use efficiency (WUE). In Arabidopsis, little is known about the variation of whole-plant water use (PWU) and whole-plant WUE (transpiration efficiency). To investigate the genetic basis of PWU, we developed a novel proxy trait by combining flowering time and rosette water use to estimate lifetime PWU. We validated its usefulness for large-scale screening of mapping populations in a subset of ecotypes. This parameter subsequently facilitated the screening of water use and drought tolerance traits in a recombinant inbred line population derived from two Arabidopsis accessions with distinct water-use strategies, namely, C24 (low PWU) and Col-0 (high PWU). Subsequent quantitative trait loci mapping and validation through near-isogenic lines identified two causal quantitative trait loci, which showed that a combination of weak and nonfunctional alleles of the FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) genes substantially reduced plant water use due to their control of flowering time. Crucially, we observed that reducing flowering time and consequently water use did not penalize reproductive performance, as such water productivity (seed produced per unit of water transpired) improved. Natural polymorphisms of FRI and FLC have previously been elucidated as key determinants of natural variation in intrinsic WUE (δ13 C). However, in the genetic backgrounds tested here, drought tolerance traits, stomatal conductance, δ13 C. and rosette water use were independent of allelic variation at FRI and FLC, suggesting that flowering is critical in determining lifetime PWU but not always leaf-level traits.

Genetic Control of Facultative Parthenocarpy in Nicotiana tabacum L.

Investigation of parthenocarpy, the production of fruit without fertilization, in multiple plant species could result in development of technologies for conferring seedless fruits and increased stability of fruit formation in economically important plants. We studied parthenocarpy in the model species Nicotiana tabacum L., and observed variability for expression of the trait among diverse genetic materials. Parthenocarpy was found to be partially dominant, and a single major quantitative trait locus on linkage group 22 was found to control the trait in a doubled haploid mapping population derived from a cross between parthenocarpic cigar tobacco cultivar "Beinhart 1000" and nonparthenocarpic flue-cured tobacco cultivar, "Hicks." The same genomic region was found to be involved with control of the trait in the important flue-cured tobacco cultivar, "K326." We also investigated the potential for the production of maternal haploids due to parthenogenesis in parthenocarpic tobacco seed capsules. Maternal haploids were not observed in parthenocarpic capsules, suggesting a requirement of fertilization for maternal haploid production due to parthenogenesis in N. tabacum.

Co-option of a default secretory pathway for plant immune responses.

Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

A regulon conserved in monocot and dicot plants defines a functional module in antifungal plant immunity.

At least two components that modulate plant resistance against the fungal powdery mildew disease are ancient and have been conserved since the time of the monocot-dicot split (≈ 200 Mya). These components are the seven transmembrane domain containing MLO/MLO2 protein and the syntaxin ROR2/PEN1, which act antagonistically and have been identified in the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana, respectively. Additionally, syntaxin-interacting N-ethylmaleimide sensitive factor adaptor protein receptor proteins (VAMP721/722 and SNAP33/34) as well as a myrosinase (PEN2) and an ABC transporter (PEN3) contribute to antifungal resistance in both barley and/or Arabidopsis. Here, we show that these genetically defined defense components share a similar set of coexpressed genes in the two plant species, comprising a statistically significant overrepresentation of gene products involved in regulation of transcription, posttranslational modification, and signaling. Most of the coexpressed Arabidopsis genes possess a common cis-regulatory element that may dictate their coordinated expression. We exploited gene coexpression to uncover numerous components in Arabidopsis involved in antifungal defense. Together, our data provide evidence for an evolutionarily conserved regulon composed of core components and clade/species-specific innovations that functions as a module in plant innate immunity.

Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant.

Arabidopsis (Arabidopsis thaliana) genes MILDEW RESISTANCE LOCUS O2 (MLO2), MLO6, and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defense responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyperaccumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady-state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single mutant and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defense against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE4/GLUCAN SYNTHASE-LIKE5 was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defense execution machinery as innate antifungal immune responses that restrict the invasion of nonadapted fungal pathogens.

Targeting Acr3 from Ensifer medicae to the plasma membrane or to the tonoplast of tobacco hairy roots allows arsenic extrusion or improved accumulation. Effect of acr3 expression on the root transcriptome.

Transgenic tobacco hairy roots expressing the bacterial arsenite efflux pump Acr3 from Ensifer medicae were generated. The gene product was targeted either to the plasma membrane (ACR3 lines) or to the tonoplast by fusing the ACR3 protein to the tonoplast integral protein TIP1.1 (TIP-ACR3 lines). Roots expressing Acr3 at the tonoplast showed greater biomass than those expressing Acr3 at the plasma membrane. Furthermore, higher contents of malondialdehyde (MDA) and RNA degradation in ACR3 lines were indicative of higher oxidative stress. The determination of ROS-scavenging enzymes depicted the transient role of peroxidases in ROS detoxification, followed by the action of superoxide dismutase during both short- and medium-term exposure periods. Regarding As accumulation, ACR3 lines accumulated up to 20-30% less As, whereas TIP-ACR3 achieved a 2-fold increase in As accumulation in comparison to control hairy roots. Strategies that presumably induce As uptake, such as phosphate deprivation or dehydration followed by rehydration in the presence of As, fostered As accumulation up to 10 800 µg g-1. Finally, the effects of the heterologous expression of acr3 on the root transcriptome were assessed. Expression at the plasma membrane induced drastic changes in gene expression, with outstanding overexpression of genes related to electron transport, ATP synthesis and ATPases, suggesting that As efflux is the main detoxification mechanism in these lines. In addition, genes encoding heat shock proteins and those related to proline synthesis and drought tolerance were activated. On the other hand, TIP-ACR3 lines showed a similar gene expression profile to that of control roots, with overexpression of the glutathione and phytochelatin synthesis pathways, together with secondary metabolism pathways as the most important resistance mechanisms in TIP-ACR3, for which As allocation into the vacuole allowed better growth and stress management. Our results suggest that modulation of As accumulation can be achieved by subcellular targeting of Acr3: expression at the tonoplast enhances As accumulation in roots, whereas expression at the plasma membrane could promote As efflux. Thus, both approaches open the possibilities for developing safer crops when grown on As-polluted paddy soils, but expression at the tonoplast leads to better growth and less stressed roots, since the high energy cost of As efflux likely compromises growth in ACR3 lines.

Natural variation of life-history traits, water use, and drought responses in Arabidopsis.

The ability of plants to acquire and use water is critical in determining life-history traits such as growth, flowering, and allocation of biomass into reproduction. In this context, a combination of functionally linked traits is essential for plants to respond to environmental changes in a coordinated fashion to maximize resource use efficiency. We analyzed different water-use traits in Arabidopsis ecotypes to identify functionally linked traits that determine water use and plant growth performance. Water-use traits measured were (i) leaf-level water-use efficiency (WUE i ) to evaluate the amount of CO 2 fixed relative to water loss per leaf area and (ii) short-term plant water use at the vegetative stage (VWU) as a measure of whole-plant transpiration. Previously observed phenotypic variance in VWU, WUE i and life-history parameters, highlighted C24 as a valuable ecotype that combined drought tolerance, preferential reproductive biomass allocation, high WUE i , and reduced water use. We therefore screened 35 Arabidopsis ecotypes for these parameters, in order to assess whether the phenotypic combinations observed in C24 existed more widely within Arabidopsis ecotypes. All parameters were measured on a short dehydration cycle. A segmented regression analysis was carried out to evaluate the plasticity of the drought response and identified the breakpoint as a reliable measure of drought sensitivity. VWU was largely dependent on rosette area, but importantly the drought sensitivity and plasticity measures were independent of the transpiring leaf surface. A breakpoint at high rSWC indicated a more drought-sensitive plant that closed stomata early during the dehydration cycle and consequently showed stronger plasticity in leaf-level WUE i parameters. None of the sensitivity, plasticity, or water-use measurements were able to predict the overall growth performance; however, there was a general trade-off between vegetative and reproductive biomass. PCA and hierarchical clustering revealed that C24 was unique among the 35 ecotypes in uniting all the beneficial water use and stress tolerance traits, while also maintaining above average plant growth. We propose that a short dehydration cycle, measuring drought sensitivity and VWU is a fast and reliable screen for plant water use and drought response strategies.

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1.

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.

A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense.

Selection pressure exerted by insects and microorganisms shapes the diversity of plant secondary metabolites. We identified a metabolic pathway for glucosinolates, known insect deterrents, that differs from the pathway activated by chewing insects. This pathway is active in living plant cells, may contribute to glucosinolate turnover, and has been recruited for broad-spectrum antifungal defense responses. The Arabidopsis CYP81F2 gene encodes a P450 monooxygenase that is essential for the pathogen-induced accumulation of 4-methoxyindol-3-ylmethylglucosinolate, which in turn is activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense. We propose that reiterated enzymatic cycles, controlling the generation of toxic molecules and their detoxification, enable the recruitment of glucosinolates in defense responses.

The Powdery Mildew Disease of Arabidopsis: A Paradigm for the Interaction between Plants and Biotrophic Fungi.

The powdery mildew diseases, caused by fungal species of the Erysiphales, have an important economic impact on a variety of plant species and have driven basic and applied research efforts in the field of phytopathology for many years. Although the first taxonomic reports on the Erysiphales date back to the 1850's, advances into the molecular biology of these fungal species have been hampered by their obligate biotrophic nature and difficulties associated with their cultivation and genetic manipulation in the laboratory. The discovery in the 1990's of a few species of powdery mildew fungi that cause disease on Arabidopsis has opened a new chapter in this research field. The great advantages of working with a model plant species have translated into remarkable progress in our understanding of these complex pathogens and their interaction with the plant host. Herein we summarize advances in the study of Arabidopsis-powdery mildew interactions and discuss their implications for the general field of plant pathology. We provide an overview of the life cycle of the pathogens on Arabidopsis and describe the structural and functional changes that occur during infection in the host and fungus in compatible and incompatible interactions, with special emphasis on defense signaling, resistance pathways, and compatibility factors. Finally, we discuss the future of powdery mildew research in anticipation of the sequencing of multiple powdery mildew genomes. The cumulative body of knowledge on powdery mildews of Arabidopsis provides a valuable tool for the study and understanding of disease associated with many other obligate biotrophic pathogen species.

A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea.

The plant cuticle composed of cutin, a lipid-derived polyester, and cuticular waxes covers the aerial portions of plants and constitutes a hydrophobic extracellular matrix layer that protects plants against environmental stresses. The botrytis-resistant 1 (bre1) mutant of Arabidopsis reveals that a permeable cuticle does not facilitate the entry of fungal pathogens in general, but surprisingly causes an arrest of invasion by Botrytis. BRE1 was identified to be long-chain acyl-CoA synthetase2 (LACS2) that has previously been shown to be involved in cuticle development and was here found to be essential for cutin biosynthesis. bre1/lacs2 has a five-fold reduction in dicarboxylic acids, the typical monomers of Arabidopsis cutin. Comparison of bre1/lacs2 with the mutants lacerata and hothead revealed that an increased permeability of the cuticle facilitates perception of putative elicitors in potato dextrose broth, leading to the presence of antifungal compound(s) at the surface of Arabidopsis plants that confer resistance to Botrytis and Sclerotinia. Arabidopsis plants with a permeable cuticle have thus an altered perception of their environment and change their physiology accordingly.

mlo-based powdery mildew immunity: silver bullet or simply non-host resistance?

SUMMARY Durability and effectiveness against all genetic variants of a microbial species are hallmarks of so-called plant 'non-host' resistance. Highly effective immunity of monocotyledonous barley against the fungal powdery mildew pathogen, which is conferred by loss-of-function mutant alleles of the barley Mlo locus, likewise is a durable and broad-spectrum type of resistance. Although this was long considered as being a barley-specific phenomenon, recent findings indicate that mlo resistance can also occur in the distantly related dicotyledonous species Arabidopsis thaliana. Shared histological and phytopathological characteristics plus a conserved requirement for a set of genes in Arabidopsis mlo and non-host powdery mildew resistance indicate a potential common mechanism for these two seemingly distinct types of immunity.