Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in banana

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato and it is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in banana. A fragment of approximately 550 bp was amplified, cloned and sequenced. The sequence analysis of several clones revealed 13 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value <3e-53), seven of them were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the banana Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs and also several conserved residues that are crucial for Pto function. Moreover, the phylogenetic analysis showed that the banana Pto-RGCs were clustered with Pto suggesting a common evolutionary origin with this R gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in banana.     

Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.     

Genetic Diversity of Pto-Like Serine/Threonine Kinase Disease Resistance Genes in Cultivated and Wild Strawberries

Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.     

The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily

Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site-leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine-threonine kinase. Southern blot analysis revealed that the pbs1-1 allele contained a deletion of the 3' end of the PBS1 open reading frame. DNA sequence analysis of the pbs1-2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition.     

A chemical-genetic approach for functional analysis of plant protein kinases

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N⁶-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases.Key words: chemical genetics, gatekeeper, LeMPK3, protein kinase, Pto, small-molecule inhibitor     

巴西橡胶Pto类抗病同源序列的克隆与系统发育重建

番茄Pto基因是一类可以编码丝氨酸/苏氨酸激酶(STK)序列的广谱抗性候选基因,其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础.在该研究中,一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列,扩增得到了一个约550 bp的基因片段,其随后被克隆并测序.序列分析发现,其中的7个抗病同源序列与Pto基因高度同源(BLASTX E value <3e-53),所以其被认为是Pto基因抗病同源序列(Pto-RGCs).通过巴西橡胶的Pto-RGCs多序列比对表明,这些序列包含了多个STKs保守的次级结构域.此外,系统发育分析也表明,巴西橡胶的Pto-RGCs属于Pto基因同源的R基因.该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源.     

Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity

Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis. Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI. However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity. In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria. Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI. The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains. However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed. The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.     

Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.     

Prf immune complexes of tomato are oligomeric and contain multiple Pto‐like kinases that diversify effector recognition

Cytoplasmic recognition of pathogen virulence effectors by plant NB‐LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB‐LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N‐terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N‐terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non‐Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.     

Two expressed soybean genes with high sequence identity to tomato Pti1 kinase lack autophosphorylation activity

An important signaling pathway for disease resistance in tomato involves the R gene product Pto which phosphorylates Ptil, a downstream member of this signaling cascade. Both Pto and Pti1 are Ser/Thr protein kinases capable of autophosphorylation in vitro. Two soybean (Glycine max L. Merr. var. Hobbit) cDNAs (sPti1a and sPti1b) were cloned and sequenced and found to each have 78% amino acid sequence identity with tomato Pti1. Glutathione S-transferase fusions of sPti1a and b expressed in Escherichia coli did not autophosphorylate in vitro, but were efficiently phosphorylated by tomato Pto. Replacement of Tyr197 with an Asp that is invariant at this position in other protein kinases did not restore autophosphorylation activity to sPti1a or b. Tyr197 was also present in the Pti1 homologues of three distant relatives of G. max. Together these results suggest that soybean Pti1 might function in a Pto-like signaling pathway that does not require Pti1 kinase activity.     

Sequence-based design of kinase inhibitors applicable for therapeutics and target identification

A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce, utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, three-dimensional structure, and involvement in protein-protein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.     

Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity

The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.     

Molecular cloning and expression of a MAP kinase homologue from pea

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.     

Adi3 is a Pdk1-interacting AGC kinase that negatively regulates plant cell death

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.     

Mammalian NDR protein kinases: from regulation to a role in centrosome duplication

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.     

The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner

In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.     

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.