Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms

Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management.     

TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes

The Toll/interleukin-1 receptor (TIR) domain is found in one of the two large families of homologues of plant disease resistance proteins (R proteins) in Arabidopsis and other dicotyledonous plants. In addition to these TIR-NBS-LRR (TNL) R proteins, we identified two families of TIR-containing proteins encoded in the Arabidopsis Col-0 genome. The TIR-X (TX) family of proteins lacks both the nucleotide-binding site (NBS) and the leucine rich repeats (LRRs) that are characteristic of the R proteins, while the TIR-NBS (TN) proteins contain much of the NBS, but lack the LRR. In Col-0, the TX family is encoded by 27 genes and three pseudogenes; the TN family is encoded by 20 genes and one pseudogene. Using massively parallel signature sequencing (MPSS), expression was detected at low levels for approximately 85% of the TN-encoding genes. Expression was detected for only approximately 40% of the TX-encoding genes, again at low levels. Physical map data and phylogenetic analysis indicated that multiple genomic duplication events have increased the numbers of TX and TN genes in Arabidopsis. Genes encoding TX, TN and TNL proteins were demonstrated in conifers; TX and TN genes are present in very low numbers in grass genomes. The expression, prevalence, and diversity of TX and TN genes suggests that these genes encode functional proteins rather than resulting from degradation or deletions of TNL genes. These TX and TN proteins could be plant analogues of small TIR-adapter proteins that function in mammalian innate immune responses such as MyD88 and Mal.     

A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.     

The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion

Plastome sequences are rich sources of information for resolving difficult phylogenetic relationships and provide genomic data for conservation studies. Here, the complete plastome sequence of Alniphyllum eberhardtii Guillaumin is reported, representing the first plastome of the basal asterid family Styracaceae (Ericales). The plastome is 155,384 bp in length and contains 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, totaling 113 unique genes with 19 genes in the inverted repeat region. Unusual features of the plastome include the presence a large 20-kb inversion in the Large Single-Copy region, the pseudogenization of the accD gene, and the loss of the second intron from clpP. The 20-kb inversion includes 14 genes and has not been previously reported in other Ericales plastomes. Thirty-nine plastid simple sequence repeats (SSRs) that may provide genetic resources for the conservation of this economically import timber plant are characterized. Phylogenetic results inferred from ML and MP analyses of 66 plastid genes and 26 taxa reveal that the Styracaceae are sister to a clade including Actinidiaceae and Ericaceae and suggest that complete plastomes are likely to be very helpful in resolving the basal relationships among Ericales families, which have resisted resolution in smaller phylogenetic data sets.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Tourist: a large family of small inverted repeat elements frequently associated with maize genes.

The wx-B2 mutation results from a 128-bp transposable element-like insertion in exon 11 of the maize Waxy gene. Surprisingly, 11 maize genes and one barley gene in the GenBank and EMBL data bases were found to contain similar elements in flanking or intron sequences. Members of this previously undescribed family of elements, designated Tourist, are short (133 bp on average), have conserved terminal inverted repeats, are flanked by a 3-bp direct repeat, and display target site specificity. Based on estimates of repetitiveness of three Tourist elements in maize genomic DNA, the copy number of the Tourist element family may exceed that of all previously reported eukaryotic inverted repeat elements. Taken together, our data suggest that Tourist may be the maize equivalent of the human Alu family of elements with respect to copy number, genomic dispersion, and the high frequency of association with genes.     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.     

Invasion of the human albumin-alpha-fetoprotein gene family by Alu, Kpn, and two novel repetitive DNA elements

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.     

Polygalacturonases, polygalacturonase-inhibiting proteins and pectic oligomers in plant-pathogen interactions

Polygalacturonases (PGs) are produced by fungal pathogens during early plant infection and are believed to be important pathogenicity factors. Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins which reduce the hydrolytic activity of endoPGs and favor the accumulation of long-chain oligogalacturonides (OGs) which are elicitors of a variety of defense responses. PGIPs belong to the superfamily of leucine reach repeat (LRR) proteins which also include the products of several plant resistance genes. A number of evidence demonstrates that PGIPs efficiently inhibit fungal invasion.     

Homology-based method for identification of protein repeats using statistical significance estimates

Short protein repeats, frequently with a length between 20 and 40 residues, represent a significant fraction of known proteins. Many repeats appear to possess high amino acid substitution rates and thus recognition of repeat homologues is highly problematic. Even if the presence of a certain repeat family is known, the exact locations and the number of repetitive units often cannot be determined using current methods. We have devised an iterative algorithm based on optimal and sub-optimal score distributions from profile analysis that estimates the significance of all repeats that are detected in a single sequence. This procedure allows the identification of homologues at alignment scores lower than the highest optimal alignment score for non-homologous sequences. The method has been used to investigate the occurrence of eleven families of repeats in Saccharomyces cerevisiae, Caenorhabditis elegans and Homo sapiens accounting for 1055, 2205 and 2320 repeats, respectively. For these examples, the method is both more sensitive and more selective than conventional homology search procedures. The method allowed the detection in the SwissProt database of more than 2000 previously unrecognised repeats belonging to the 11 families. In addition, the method was used to merge several repeat families that previously were supposed to be distinct, indicating common phylogenetic origins for these families.     

Expansion of protein domain repeats

Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.     

Plant disease resistance protein signaling: NBS-LRR proteins and their partners

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.     

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.     

Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi

Polygalacturonase-inhibiting proteins (PGIPs) are ubiquitous plant cell wall proteins that are directed against fungal polygalacturonases (PGs), which are important pathogenicity factors. The inhibiting activity of PGIPs directly reduces the aggressive potential of PGs. In addition, it causes PGs to form more long-chain oligogalacturonides that are able to induce defense responses, thereby indirectly contributing to the plant defense. Recent evidence demonstrates that PGIPs are efficient defense proteins and limit fungal invasion. PGIPs and the products of many plant resistance genes share a leucine-rich repeat (LRR) structure, which provides specific recognition of pathogen-derived molecules. The high level of polymorphism of both PGIPs and polygalacturonases is an invaluable tool for deciphering the structure, function and evolution of plant LRR proteins and their ligands. Furthermore, information about PGIP structure and evolution paves the way to the development of efficient strategies for crop protection.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Polygalacturonase inhibiting proteins: players in plant innate immunity?

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular leucine-rich repeat (LRR) proteins that recognize and inhibit fungal polygalacturonases (PGs). The PG-PGIP interaction favours the accumulation of elicitor-active oligogalacturonides and causes the activation of defence responses. Small gene families encode PGIP isoforms that differ in affinity and specificity for PGs secreted by different pathogens. The consensus motif within the LRR structure of PGIPs is the same as that of the extracellular receptors of the plant innate immune system. Structural and functional evidence suggest that PGIPs are versatile proteins involved in innate immunity and that they are capable of recognizing different surface motifs of functionally related but structurally variable PGs.