The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination.

Gibberellic acid (GA)-mediated cell expansion initiates the seed-to-seedling transition in plants and is repressed by DELLA proteins. Using digital single-cell analysis, we identified a cellular subdomain within the midhypocotyl, whose expansion drives the final step of this developmental transition under optimal conditions. Using network inference, the transcription factor ATHB5 was identified as a genetic factor whose localized expression promotes GA-mediated expansion specifically within these cells. Both this protein and its putative growth-promoting target EXPANSIN3 are repressed by DELLA, and coregulated at single-cell resolution during seed germination. The cellular domains of hormone sensitivity were explored within the Arabidopsis (Arabidopsis thaliana) embryo by putting seeds under GA-limiting conditions and quantifying cellular growth responses. The middle and upper hypocotyl have a greater requirement for GA to promote cell expansion than the lower embryo axis. Under these conditions, germination was still completed following enhanced growth within the radicle and lower axis. Under GA-limiting conditions, the athb5 mutant did not show a phenotype at the level of seed germination, but it did at a cellular level with reduced cell expansion in the hypocotyl relative to the wild type. These data reveal that the spatiotemporal cell expansion events driving this transition are not determinate, and the conditional use of GA-ATHB5-mediated hypocotyl growth under optimal conditions may be used to optionally support rapid seedling growth. This study demonstrates that multiple genetic and spatiotemporal cell expansion mechanisms underlie the seed to seedling transition in Arabidopsis.

Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using Fluorescence Activated Cell Sorting.

Changes in gene expression form a crucial part of the plant response to infection. In the last decade, whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, with some pathogens such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response encompassing responses of non-infected as well as infected cells within the leaf. Highly localized expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells. Furthermore, local and systemic Hpa responses of a differing nature may become conflated. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. We isolated haustoriated (Hpa-proximal) and non-haustoriated (Hpa-distal) cells from infected seedling samples using FACS, and measured global gene expression. When compared with an uninfected control, 278 transcripts were identified as significantly differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many highly locally regulated genes that can be implicated as novel in the Hpa response, and that were uncovered for the first time using our sensitive FACS technique.

Improving crop disease resistance: lessons from research on Arabidopsis and tomato.

One of the great challenges for food security in the 21st century is to improve yield stability through the development of disease-resistant crops. Crop research is often hindered by the lack of molecular tools, growth logistics, generation time and detailed genetic annotations, hence the power of model plant species. Our knowledge of plant immunity today has been largely shaped by the use of models, specifically through the use of mutants. We examine the importance of Arabidopsis and tomato as models in the study of plant immunity and how they help us in revealing a detailed and deep understanding of the various layers contributing to the immune system. Here we describe examples of how knowledge from models can be transferred to economically important crops resulting in new tools to enable and accelerate classical plant breeding. We will also discuss how models, and specifically transcriptomics and effectoromics approaches, have contributed to the identification of core components of the defense response which will be key to future engineering of durable and sustainable disease resistance in plants.

A growth quantification assay for Hyaloperonospora arabidopsidis isolates in Arabidopsis thaliana.

There is a considerable interest in determining the role of individual oomycete effectors in promoting disease. Widely used strategies are based on manipulating effector-expression levels in the pathogen and by over-expressing particular effectors in the host by genetic transformation. In the case of the oomycete, Hyaloperonospora arabidopsidis (Hpa) genetic manipulation is not yet possible, so over-expression of predicted effectors in stably transformed Arabidopsis lines is used to investigate their capability for promoting virulence. Here, we describe a technique for quantifying pathogen growth based on the counting of asexual reproductive structures called sporangiophores in the compatible interaction between the Hpa isolate Noks1 and the Col-0 Arabidopsis accession.

A simple and fast protocol for the protein complex immunoprecipitation (Co-IP) of effector: host protein complexes.

Plant pathogens are responsible for enormous damage in natural and cultured ecosystems. One strategy most pathogenic organisms follow is the secretion of effector proteins that manipulate the host immune system to suppress defense responses. There is considerable interest in finding host targets of pathogen effectors as this helps to shape our understanding of how those proteins work in planta. The presented protocol describes a protein complex immunoprecipitation method aimed at verifying protein-protein interactions derived from protein complementation assays like Yeast-two-Hybrid.

An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.

The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection.

Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H2O2 signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.

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.

Maintenance of genetic variation in plants and pathogens involves complex networks of gene-for-gene interactions.

The RPP13 [recognition of Hyaloperonospora arabidopsidis (previously known as Peronospora parasitica)] resistance (R) gene in Arabidopsis thaliana exhibits the highest reported level of sequence diversity among known R genes. Consistent with a co-evolutionary model, the matching effector protein ATR13 (A. thaliana-recognized) from H. arabidopsidis reveals extreme levels of allelic diversity. We isolated 23 new RPP13 sequences from a UK metapopulation, giving a total of 47 when combined with previous studies. We used these in functional studies of the A. thaliana accessions for their resistance response to 16 isolates of H. arabidopsidis. We characterized the molecular basis of recognition by the expression of the corresponding ATR13 genes from these 16 isolates in these host accessions. This allowed the determination of which alleles of RPP13 were responsible for pathogen recognition and whether recognition was dependent on the RPP13/ATR13 combination. Linking our functional studies with phylogenetic analysis, we determined that: (i) the recognition of ATR13 is mediated by alleles in just a single RPP13 clade; (ii) RPP13 alleles in other clades have evolved the ability to detect other pathogen ATR protein(s); and (iii) at least one gene, unlinked to RPP13 in A. thaliana, detects a different subgroup of ATR13 alleles.

Natural variation reveals key amino acids in a downy mildew effector that alters recognition specificity by an Arabidopsis resistance gene.

RPP13, a member of the cytoplasmic class of disease resistance genes, encodes one of the most variable Arabidopsis proteins so far identified. This variability is matched in ATR13, the protein from the oomycete downy mildew pathogen Hyaloperonospora parasitica recognized by RPP13, suggesting that these proteins are involved in tight reciprocal coevolution. ATR13 exhibits five domains: an N-terminal signal peptide, an RXLR motif, a heptad leucine/isoleucine repeat, an 11-amino-acid repeated sequence and a C-terminal domain. We show that the conserved RXLR-containing domain is dispensable for ATR13-mediated recognition, consistent with its role in transport into the plant cytoplasm. Sequencing ATR13 from 16 isolates of H. parasitica revealed high levels of amino acid diversity across the entire protein. The leucines/isoleucines of the heptad leucine repeat were conserved, and mutation of particular leucine or isoleucine residues altered recognition by RPP13. Natural variation has not exploited this route to detection avoidance, suggesting a key role of this domain in pathogenicity. The extensive variation in the 11-amino-acid repeat units did not affect RPP13 recognition. Domain swap analysis showed that recognition specificity lay in the C-terminal domain of ATR13. Variation analyses combined with functional assays allowed the identification of four amino acid positions that may play a role in recognition specificity. Site-directed mutagenesis confirmed that a threonine residue is absolutely required for RPP13 recognition and that recognition can be modulated by the presence of either an arginine or glutamic acid at other sites. Mutations in these three amino acids had no effect on the interaction of ATR13 with a resistance gene unlinked to RPP13, consistent with their critical role in determining RPP13-Nd recognition specificity.

Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

Trafficking arms: oomycete effectors enter host plant cells.

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.

An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm.

The oomycete Phytophthora infestans causes late blight, the potato disease that precipitated the Irish famines in 1846 and 1847. It represents a reemerging threat to potato production and is one of >70 species that are arguably the most devastating pathogens of dicotyledonous plants. Nevertheless, little is known about the molecular bases of pathogenicity in these algae-like organisms or of avirulence molecules that are perceived by host defenses. Disease resistance alleles, products of which recognize corresponding avirulence molecules in the pathogen, have been introgressed into the cultivated potato from a wild species, Solanum demissum, and R1 and R3a have been identified. We used association genetics to identify Avr3a and show that it encodes a protein that is recognized in the host cytoplasm, where it triggers R3a-dependent cell death. Avr3a resides in a region of the P. infestans genome that is colinear with the locus containing avirulence gene ATR1(NdWsB) in Hyaloperonospora parasitica, an oomycete pathogen of Arabidopsis. Remarkably, distances between conserved genes in these avirulence loci were often similar, despite intervening genomic variation. We suggest that Avr3a has undergone gene duplication and that an allele evading recognition by R3a arose under positive selection.

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines.

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.

Host-parasite coevolutionary conflict between Arabidopsis and downy mildew.

Plants are constantly exposed to attack by an array of diverse pathogens but lack a somatically adaptive immune system. In spite of this, natural plant populations do not often suffer destructive disease epidemics. Elucidating how allelic diversity within plant genes that function to detect pathogens (resistance genes) counteracts changing structures of pathogen genes required for host invasion (pathogenicity effectors) is critical to our understanding of the dynamics of natural plant populations. The RPP13 resistance gene is the most polymorphic gene analyzed to date in the model plant Arabidopsis thaliana. Here we report the cloning of the avirulence gene, ATR13, that triggers RPP13-mediated resistance, and we show that it too exhibits extreme levels of amino acid polymorphism. Evidence of diversifying selection visible in both components suggests that the host and pathogen may be locked in a coevolutionary conflict at these loci, where attempts to evade host resistance by the pathogen are matched by the development of new detection capabilities by the host.

The arabidopsis TIR-NB-LRR gene RAC1 confers resistance to Albugo candida (white rust) and is dependent on EDS1 but not PAD4.

Resistance to Albugo candida isolate Acem1 is conferred by a dominant gene, RAC1, in accession Ksk-1 of Arabidopsis thaliana. This gene was isolated by positional cloning and is a member of the Drosophila toll and mammalian interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NB-LRR) class of plant resistance genes. Strong identity of the TIR and NB domains was observed between the predicted proteins encoded by the Ksk-1 allele and the allele from an Acem1-susceptible accession Columbia (Col) (99 and 98%, respectively). However, major differences between the two predicted proteins occur within the LRR domain and mainly are confined to the beta-strand/beta-turn structure of the LRR. Both proteins contain 14 imperfect repeats. RAC1-mediated resistance was analyzed further using mutations in defense regulation, including: pad4-1, eds1-1, and NahG, in the presence of the RAC1 allele from Ksk-1. White rust resistance was completely abolished by eds1-1 but was not affected by either pad4-1 or NahG.

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana.

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.

A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd.

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the Arabidopsis thaliana-recognised (ATR) avirulence genes. We describe the identification of 10 amplified fragment length polymorphism (AFLP) markers that define a genetic mapping interval for the ATR1Nd avirulence allele, the presence of which is perceived by the RPP1Nd resistance gene. Furthermore, we have constructed a P. parasitica (At) bacterial artificial chromosome (BAC) library comprising over 630Mb of cloned DNA. We have isolated 16 overlapping clones from the BAC library that form a contig spanning the genetic interval. BAC sequence-derived markers and a total mapping population of 311 F(2) individuals were used to refine the ATR1Nd locus to a 1cM interval that is represented by four BAC clones and spans less than 250kb of DNA. This work demonstrates that map-based cloning techniques are feasible in this organism and provides the critical foundations for cloning ATR1Nd using such a strategy.

Use of suppression subtractive hybridization to identify downy mildew genes expressed during infection of Arabidopsis thaliana.

SUMMARY Peronospora parasitica is an obligate biotrophic oomycete that causes downy mildew in Arabidopsis thaliana and Brassica species. Our goal is to identify P. parasitica (At) genes that are involved in pathogenicity. We used suppression subtractive hybridization (SSH) to generate cDNA libraries enriched for in planta-expressed parasite genes and up-regulated host genes. A total of 1345 clones were sequenced representing cDNA fragments from 25 putative P. parasitica (At) genes (Ppat 1-25) and 618 Arabidopsis genes. Analyses of expression patterns showed that 15 Ppats were expressed only in planta. Eleven Ppats encoded peptides with homology (BlastP values < 1e-05) to proteins with roles in membrane or cell wall biosynthesis, amino acid metabolism, osmoregulation, cation transport, phosphorylation or protein secretion. The other 14 represent potentially novel oomycete genes with none having homologues in an extensive Phytophthora species EST database. A full-length sequence was obtained for four Ppats and each encoded small cysteine-rich proteins with amino-terminal signal peptide sequences. These results demonstrate the utility of SSH in obtaining novel in planta-expressed genes from P. parasitica (At) that complements other gene discovery approaches such as EST sequencing.