CITRX thioredoxin is a putative adaptor protein connecting Cf-9 and the ACIK1 protein kinase during the Cf-9/Avr9- induced defence response

Tomato Cf-9, a receptor-like protein (RLP), confers resistance to races of the fungal pathogen Cladosporium fulvum that express the Avr9 avirulence gene. CITRX (Cf-9-interacting thioredoxin) was previously identified in a yeast two-hybrid screen as a protein interacting with the cytoplasmic domain of Cf-9 and shown to be a negative regulator of the cell death induced after Cf-9/Avr9 interaction. ACIK1 is a Ser/Thr protein kinase that is specifically required for the Cf-9 and Cf-4 dependent defence response in tomato. In this paper we present data suggesting that CITRX may act as an adaptor recruiting the ACIK1 kinase to the cytoplasmic domain of Cf-9 upon elicitation with the Avr9 peptide. Interestingly, the catalytic activities of both CITRX and ACIK1 are not required for their interaction.     

Rearrangements in the Cf-9 disease resistance gene cluster of wild tomato have resulted in three genes that mediate Avr9 responsiveness

Cf resistance genes in tomato confer resistance to the fungal leaf pathogen Cladosporium fulvum. Both the well-characterized resistance gene Cf-9 and the related 9DC gene confer resistance to strains of C. fulvum that secrete the Avr9 protein and originate from the wild tomato species Lycopersicon pimpinellifolium. We show that 9DC and Cf-9 are allelic, and we have isolated and sequenced the complete 9DC cluster of L. pimpinellifolium LA1301. This 9DC cluster harbors five full-length Cf homologs, including orthologs of the most distal homologs of the Cf-9 cluster and three central 9DC genes. Two 9DC genes (9DC1 and 9DC2) have an identical coding sequence, whereas 9DC3 differs at its 3' terminus. From a detailed comparison of the 9DC and Cf-9 clusters, we conclude that the Cf-9 and Hcr9-9D genes from the Cf-9 cluster are ancestral to the first 9DC gene and that the three 9DC genes were generated by subsequent intra- and intergenic unequal recombination events. Thus, the 9DC cluster has undergone substantial rearrangements in the central region, but not at the ends. Using transient transformation assays, we show that all three 9DC genes confer Avr9 responsiveness, but that 9DC2 is likely the main determinant of Avr9 recognition in LA1301.     

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.     

Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein Avr4

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.     

RFLP linkage analysis of the Cf-4 and Cf-9 genes for resistance toCladosporium fulvum in tomato

Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F₂ progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.     

Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies sequences that determine recognitional specificity in Cf-4 and Cf-9.

In many interactions between plants and their pathogens, resistance to infection is specified by plant resistance (R) genes and corresponding pathogen avirulence (Avr) genes. In tomato, the Cf-4 and Cf-9 resistance genes map to the same location but confer resistance to Cladosporium fulvum through recognition of different avirulence determinants (AVR4 and AVR9) by a molecular mechanism that has yet to be determined. Here, we describe the cloning and characterization of Cf-4, which also encodes a membrane-anchored extracellular glycoprotein. Cf-4 contains 25 leucine-rich repeats, which is two fewer than Cf-9. The proteins have > 91% identical amino acids. DNA sequence comparison suggests that Cf-4 and Cf-9 are derived from a common progenitor sequence. Amino acid differences distinguishing Cf-4 and Cf-9 are confined to their N termini, delimiting a region that determines the recognitional specificity of ligand binding. The majority of these differences are in residues interstitial to those of the leucine-rich repeat consensus motif. Many of these residues are predicted to form a solvent-exposed surface that can interact with the cognate ligand. Both Cf-4 and Cf-9 are located within a 36-kb region comprising five tandemly duplicated homologous genes. These results provide further insight into the molecular basis of pathogen perception by plants and the organization of complex R gene loci.     

The Cf-ECP2 gene is linked to, but not part of, the Cf-4/Cf-9 cluster on the short arm of chromosome 1 in tomato

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F₂ and F₃ populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster “Milky Way”. Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus. Received: 15 June 1999 / Accepted: 24 August 1999     

Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility

The key regulator of salicylic acid (SA)-mediated resistance, NPR1, is functionally conserved in diverse plant species, including rice (Oryza sativa L.). Investigation in depth is needed to provide an understanding of NPR1-mediated resistance and a practical strategy for the improvement of disease resistance in the model crop rice. The rice genome contains five NPR1-like genes. In our study, three rice homologous genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, were found to be induced by rice bacterial blight Xanthomonas oryzae pv. oryzae and rice blast Magnaporthe grisea, and the defence molecules benzothiadiazole, methyl jasmonate and ethylene. We confirmed that OsNPR1 is the rice orthologue by complementing the Arabidopsis npr1 mutant. Over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants. The OsNPR1-green fluorescent protein (GFP) fusion protein was localized in the cytoplasm and moved into the nucleus after redox change. Mutations in its conserved cysteine residues led to the constitutive localization of OsNPR1(2CA)-GFP in the nucleus and also abolished herbivore hypersensitivity in transgenic rice. Different subcellular localizations of OsNPR1 antagonistically regulated SA- and jasmonic acid (JA)-responsive genes, but not SA and JA levels, indicating that OsNPR1 might mediate antagonistic cross-talk between the SA- and JA-dependent pathways in rice. This study demonstrates that rice has evolved an SA-mediated systemic acquired resistance similar to that in Arabidopsis, and also provides a practical approach for the improvement of disease resistance without the penalty of decreased herbivore resistance in rice.     

The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells

A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.     

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

Background

Plants have two related immune systems to defend themselves against pathogen attack. Initially, pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses.

Results

We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling.

Conclusions

Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.     

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

Mutational analysis of Cak1p, an essential protein kinase that regulates cell cycle progression

In Saccharomyces cerevisiae, entry into S phase requires the activation of the protein kinase Cdc28p through binding with cyclin Clb5p or Clb6p, as well as the destruction of the cyclin-dependent kinase inhibitor Sic1p. Mutants that are defective in this activation event arrest after START, with unreplicated DNA and multiple, elongated buds. These mutants include cells defective in CDC4, CDC34 or CDC53, as well as cells that have lost all CLB function. Here we describe mutations in another gene, CAK1, that lead to a similar arrest. Cells that are defective in CAK1 are inviable and arrest with a single nucleus and multiple, elongated buds. CAK1 encodes a protein kinase most closely related to the Cdc2p family of protein kinases. Mutations that lead to the production of an inactive kinase that can neither autophosphorylate, nor phosphorylate Cdc28p in vitro are also incapable of rescuing a cell with a deletion of CAK1. These results underscore the importance of the Cak1p protein kinase activity in cell cycle progression. Received: 2 January 1997 / Accepted: 20 June 1997     

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.