DELLA proteins positively regulate seed size in Arabidopsis.

Human and animal nutrition is mainly based on seeds. Seed size is a key factor affecting seed yield and has thus been one of the primary objectives of plant breeders since the domestication of crop plants. Seed size is coordinately regulated by signals of maternal and zygotic tissues that control the growth of the seed coat, endosperm and embryo. Here, we provide previously unreported evidence for the role of DELLA proteins, key repressors of gibberellin responses, in the maternal control of seed size. The gain-of-function della mutant gai-1 produces larger seeds as a result of an increase in the cell number in ovule integuments. This leads to an increase in ovule size and, in turn, to an increase in seed size. Moreover, DELLA activity promotes increased seed size by inducing the transcriptional activation of AINTEGUMENTA, a genetic factor that controls cell proliferation and organ growth, in the ovule integuments of gai-1. Overall, our results indicate that DELLA proteins are involved in the control of seed size and suggest that modulation of the DELLA-dependent pathway could be used to improve crop yield.

Gibberellins regulate ovule number through a DELLA-CUC2 complex in Arabidopsis.

Ovule development is a key process for plant reproduction, helping to ensure correct seed production. Several molecular factors and plant hormones such as gibberellins are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been shown to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA acts in ovule primordia initiation remained unknown. In this study we report that DELLA proteins participate in ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, through the direct GAI-CUC2 interaction in placental cells that would determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and co-localization in the placenta supports this hypothesis. Moreover, molecular analysis identified a subset of the loci for which the GAI protein may act as a transcriptional co-regulator in a CUC2-dependent manner. The DELLA-CUC2 complex is a component of the gene regulatory network controlling ovule primordia initiation in Arabidopsis.

A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1.

KEY MESSAGE: The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar 'Piel de Sapo' (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms.

Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.

Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis.

Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1Δ17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1Δ17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1Δ17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1Δ17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development.

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity.

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator.

Non-recognition-of-BTH4, an Arabidopsis mediator subunit homolog, is necessary for development and response to salicylic acid.

Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana non-expresser of pathogenesis-related gene 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.

Specific missense alleles of the arabidopsis jasmonic acid co-receptor COI1 regulate innate immune receptor accumulation and function.

Plants utilize proteins containing nucleotide binding site (NB) and leucine-rich repeat (LRR) domains as intracellular innate immune receptors to recognize pathogens and initiate defense responses. Since mis-activation of defense responses can lead to tissue damage and even developmental arrest, proper regulation of NB-LRR protein signaling is critical. RAR1, SGT1, and HSP90 act as regulatory chaperones of pre-activation NB-LRR steady-state proteins. We extended our analysis of mutants derived from a rar1 suppressor screen and present two allelic rar1 suppressor (rsp) mutations of Arabidopsis COI1. Like all other coi1 mutations, coi1(rsp) missense mutations impair Jasmonic Acid (JA) signaling resulting in JA-insensitivity. However, unlike previously identified coi1 alleles, both coi1(rsp) alleles lack a male sterile phenotype. The coi1(rsp) mutants express two sets of disease resistance phenotypes. The first, also observed in coi1-1 null allele, includes enhanced basal defense against the virulent bacterial pathogen Pto DC3000 and enhanced effector-triggered immunity (ETI) mediated by the NB-LRR RPM1 protein in both rar1 and wild-type backgrounds. These enhanced disease resistance phenotypes depend on the JA signaling function of COI1. Additionally, the coi1(rsp) mutants showed a unique inability to properly regulate RPM1 accumulation and HR, exhibited increased RPM1 levels in rar1, and weakened RPM1-mediated HR in RAR1. Importantly, there was no change in the steady-state levels or HR function of RPM1 in coi1-1. These results suggest that the coi1(rsp) proteins regulate NB-LRR protein accumulation independent of JA signaling. Based on the phenotypic similarities and genetic interactions among coi1(rsp), sgt1b, and hsp90.2(rsp) mutants, our data suggest that COI1 affects NB-LRR accumulation via two NB-LRR co-chaperones, SGT1b and HSP90. Together, our data demonstrate a role for COI1 in disease resistance independent of JA signaling and provide a molecular link between the JA and NB-LRR signaling pathways.

The BLADE-ON-PETIOLE genes of Arabidopsis are essential for resistance induced by methyl jasmonate.

BACKGROUND: NPR1 is a gene of Arabidopsis thaliana required for the perception of salicylic acid. This perception triggers a defense response and negatively regulates the perception of jasmonates. Surprisingly, the application of methyl jasmonate also induces resistance, and NPR1 is also suspected to be relevant. Since an allelic series of npr1 was recently described, the behavior of these alleles was tested in response to methyl jasmonate. RESULTS: The response to methyl jasmonate of different npr1s alleles and NPR1 paralogs null mutants was measured by the growth of a pathogen. We have also tested the subcellular localization of some npr1s, along with the protein-protein interactions that can be measured in yeast. The localization of the protein in npr1 alleles does not affect the response to methyl jasmonate. In fact, NPR1 is not required. The genes that are required in a redundant fashion are the BOPs. The BOPs are paralogs of NPR1, and they physically interact with the TGA family of transcription factors. CONCLUSIONS: Some npr1 alleles have a phenotype in this response likely because they are affecting the interaction between BOPs and TGAs, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants.

Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation.

Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open-close cycling of a known "lid structure" that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome.

Quantitative genetic analysis of salicylic acid perception in Arabidopsis.

Salicylic acid (SA) is a phytohormone required for a full resistance against some pathogens in Arabidopsis, and NPR1 (Non-Expressor of Pathogenesis Related Genes 1) is the only gene with a strong effect on resistance induced by SA which has been described. There can be additional components of SA perception that escape the traditional approach of mutagenesis. An alternative to that approach is searching in the natural variation of Arabidopsis. Different methods of analyzing the variation between ecotypes have been tried and it has been found that measuring the growth of a virulent isolate of Pseudomonas syringae after the exogenous application of SA is the most effective one. Two ecotypes, Edi-0 and Stw-0, have been crossed, and their F2 has been studied. There are two significant quantitative trait loci (QTLs) in this population, and there is one QTL in each one of the existing mapping populations Col-4 × Laer-0 and Laer-0 × No-0. They have different characteristics: while one QTL is only detectable at low concentrations of SA, the other acts after the point of crosstalk with methyl jasmonate signalling. Three of the QTLs have candidates described in SA perception as NPR1, its interactors, and a calmodulin binding protein.

Resistance and biomass in Arabidopsis: a new model for salicylic acid perception.

Salicylic acid (SA) is an essential hormone for plant defence and development. SA perception is usually measured by counting the number of pathogens that grow in planta upon an exogenous application of the hormone. A biological SA perception model based on plant fresh weight reduction caused by disease resistance in Arabidopsis thaliana is proposed. This effect is more noticeable when a chemical analogue of SA is used, like Benzothiadiazole (BTH). By spraying BTH several times, a substantial difference in plant biomass is observed when compared with the mock treatment. Such difference is dose-dependent and does not require pathogen inoculation. The model is robust and allows for the comparison of different Arabidopsis ecotypes, recombinant inbreed lines, and mutants. Our results show that two mutants, non-expresser of pathogenesis-related genes 1 (npr1) and auxin resistant 3 (axr3), fail to lose biomass when BTH is applied to them. Further experiments show that axr3 responds to SA and BTH in terms of defence induction. NPR1-related genotypes also confirm the pivotal role of NPR1 in SA perception, and suggest an active program of depletion of resources in the infected tissues.

The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.

*The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

Structure-function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid.

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild-type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high-throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl-terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis.

Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.

NPR1 paralogs of Arabidopsis and their role in salicylic acid perception.

Salicylic acid (SA) is responsible for certain plant defence responses and NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) is the master regulator of SA perception. In Arabidopsis thaliana there are five paralogs of NPR1. In this work we tested the role of these paralogs in SA perception by generating combinations of mutants and transgenics. NPR2 was the only paralog able to partially complement an npr1 mutant. The null npr2 reduces SA perception in combination with npr1 or other paralogs. NPR2 and NPR1 interacted in all the conditions tested, and NPR2 also interacted with other SA-related proteins as NPR1 does. The remaining paralogs behaved differently in SA perception, depending on the genetic background, and the expression of some of the genes induced by SA in an npr1 background was affected by the presence of the paralogs. NPR2 fits all the requirements of an SA receptor while the remaining paralogs also work as SA receptors with a strong hierarchy. According to the data presented here, the closer the gene is to NPR1, the more relevant its role in SA perception.

ß-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis.

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, ßCA1 and ßCA2, are ß-carbonic anhydrase family proteins. Since double mutant ßca1 ßca2 plants did not show any obvious phenotype, we investigated other ßCAs and found that NRB4 also interacts with ßCA3 and ßCA4. Moreover, several ßCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between ßCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4-ßCA1-NPR1. The quintuple mutant ßca1 ßca2 ßca3 ßca4 ßca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.

NHL25 and NHL3, two NDR1/HIN1-1ike genes in Arabidopsis thaliana with potential role(s) in plant defense.

The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene. Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation. Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv. tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4. Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation. However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced. Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3. Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected. This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes. Bacteria-induced gene expression was not abolished in ethylene- (etrl-3 and ein2-1) and jasmonate- (coil-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1). Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv. tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv. tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis. Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance.

An allele of Arabidopsis COI1 with hypo- and hypermorphic phenotypes in plant growth, defence and fertility.

Resistance to biotrophic pathogens is largely dependent on the hormone salicylic acid (SA) while jasmonic acid (JA) regulates resistance against necrotrophs. JA negatively regulates SA and is, in itself, negatively regulated by SA. A key component of the JA signal transduction pathway is its receptor, the COI1 gene. Mutations in this gene can affect all the JA phenotypes, whereas mutations in other genes, either in JA signal transduction or in JA biosynthesis, lack this general effect. To identify components of the part of the resistance against biotrophs independent of SA, a mutagenised population of NahG plants (severely depleted of SA) was screened for suppression of susceptibility. The screen resulted in the identification of intragenic and extragenic suppressors, and the results presented here correspond to the characterization of one extragenic suppressor, coi1-40. coi1-40 is quite different from previously described coi1 alleles, and it represents a strategy for enhancing resistance to biotrophs with low levels of SA, likely suppressing NahG by increasing the perception to the remaining SA. The phenotypes of coi1-40 lead us to speculate about a modular function for COI1, since we have recovered a mutation in COI1 which has a number of JA-related phenotypes reduced while others are equal to or above wild type levels.

Identification of new type III effectors and analysis of the plant response by competitive index.

In recent years, many efforts have been directed towards the identification of new type III-secreted effectors, and the completion of the secretomes of several Pseudomonas syringae pathovars. Several functional and bioinformatic screenings have been used to search for candidates, which have been tested for translocation into the plant cell, an essential criterion for the identification of new type III effector proteins. The most common translocation assay is based on the use of DeltaAvrRpt2 as a reporter. When fused to a type III effector protein, DeltaAvrRpt2 is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 resistance protein. This approach has been used widely and has allowed the identification of a considerable number of new effectors in a fast and convenient manner. However, as the hypersensitive response is a semi-quantitative assay, and the conditions do not resemble those occurring in nature, effectors with low expression or translocation efficiency could fail to translocate sufficient DeltaAvrRpt2 to trigger a clear hypersensitive response. In keeping with these limitations, this test has failed to detect some true effectors that have been confirmed as such by other means. In order to increase the sensitivity of this method, we have developed a modification of the DeltaAvrRpt2-based translocation assay using a competitive index in mixed infection to monitor the limitation of growth associated with the induction of the hypersensitive response. We have tested several effector candidates from P. syringae pv. phaseolicola and other P. syringae pathovars, and have compared the results obtained by our competitive index translocation assay with those obtained by standard hypersensitive response assays. We have identified six type III secretion system-translocated proteins using this approach, five of which failed to be identified by hypersensitive response assays. In addition, we have analysed the defence response triggered by one of these effectors using competitive index assays.