Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

PD1, an S-like RNase gene from a self-incompatible cultivar of almond

 Many flowering plants contain stylar S-RNases that are involved in self-incompatibility and S-like RNases of which the biological function is uncertain. This paper reports the deduced amino acid sequence of an S-like RNase gene (PD1) from the self-incompatible plant Prunus dulcis (almond). The amino acid sequence of PD1, which was derived from cDNA and genomic DNA clones, showed 34–86% identity to acidic plant S-like RNases reported so far, with the highest degree of similarity being to an S-like RNase from Japanese pear (Pyrus pyrifolia). Based on RNA hybridisation experiments it appears that, like for many other S-like RNases, the expression of PD1 is not pistil-specific. Analysis of the genomic structure revealed the presence of three introns, of which one is similar in location to that of the related S-RNase gene from Solanaceae and Rosaceae. At least four bands hybridising to PD1 were found upon Southern hybridisation, suggesting the presence of a multigene family of S-like RNase genes in almond. The putative biological function of PD1 is discussed. Received: 22 November 1999 / Revision received: 18 February 2000 · Accepted: 13 March 2000     

S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.     

Identification and evolutionary analysis of a relic S-RNase in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

In several gametophytic self-incompatible species of the Solanaceae, a group of RNases named relic S-RNase has been identified that belong to the S-RNase lineage but are no longer involved in self-incompatibility. However, their function, evolution and presence in the Scrophulariaceae remained largely unknown. Here, we analyzed the expression of S-RNase and its related genes in Antirrhinum, a member of the Scrophulariacaeae, and identified a pistil-specific RNase gene; AhRNase29 encodes a predicted polypeptide of 235 amino acids with an estimated molecular weight of 26 kDa. Sequence and phylogenetic analyses indicated that AhRNase29 forms a monophyletic clade with Antirrhinum S-RNases, similar to that observed for other relic S-RNases. Possible evolution and function of relic S-RNases are discussed.     

A barley gene (rsh1) encoding a ribonuclease S-like homologue specifically expressed in young light-grown leaves

 A group of frequent cDNA clones from a young-leaf cDNA library was found to code for a homologue of S-ribonucleases (S-RNases) involved in gametophytic incompatibility and the so-called S-like RNases active in flowers and in vegetative tissues. The derived amino acid sequence starts with a signal peptide and has a 27-amino-acid C-terminal extension of unknown function. The barley (Hordeum vulgare L.) gene, rsh1 (for RNase S-like homologue) corresponding to the cDNA clones was isolated. The gene has three introns and the position of one intron corresponds to the site of the single, small intron in the S-RNase genes. The deduced amino acid sequence of mature RSH1 shares 35% identical and 58% similar amino acid residues with an S-like RNase from tomato, RNase LE. However, two active-site histidine residues, conserved between all S and S-like RNases are replaced by serine residues in RSH1. The new barley RNase S-like homologue is clearly related to the family of active RNases but is probably not active as an RNase. Sequences from the same class of presumably inactive RNases have been recorded in maize, rice and sorghum. The barley gene is exclusively expressed in young leaf tissue and is substantially induced by light. Received: 26 July 1999 / Accepted: 26 October 1999     

Self-incompatibility RNases from three plant families: homology or convergence?

In the Rosaceae, Scrophulariaceae, and Solanaceae, the stylar product of the self-incompatibility (S-) locus is an RNase. Using protein sequence data from 34 RNase genes (three fungal RNases, seven angiosperm non-S RNases, 11 Rosaceae S-alleles, three Scrophulariaceae S-alleles, and ten Solanaceae S-alleles) we reconstructed the genealogy of angiosperm RNases using the neighbor joining method and two distance metrics in order to assess whether use of S-RNases in these families is the result of homology or convergence. Four monophyletic groups of angiosperm RNases were found: the S-RNases of each of the three families and a group comprising most of the angiosperm non-S RNases. The S-RNases of the Scrophulariaceae and Solanaceae were found to be homologous but strong inference concerning the homology or convergence of S-RNases from the Rosaceae with those of the other families was not possible because of uncertain placement of both the root and two of the angiosperm non-S RNases. The most recent common ancestor of the Rosaceae and both the Scrophulariaceae and Solanaceae is shared by ~80% of dicot families. If the -RNases of the Rosaceae are homologous to those of the Scrophulariaceae and Solanaceae, then many other dicot families might be expected to share RNases as the mechanism of gametophytic self-incompatibility.     

A tobacco S-like RNase inhibits hyphal elongation of plant pathogens

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.     

Structural and functional characteristics of S-like ribonucleases from carnivorous plants

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k _cat/K _m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k _cat/K _m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k _cat/K _m values or k _cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.     

The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories

The RNase A superfamily has been important in biochemical, structural, and evolutionary studies and is believed to be the sole vertebrate-specific enzyme family. To understand the origin and diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of human, mouse, rat, and chicken. We report a previously unnoticed gene cluster in mouse chromosome 10 and a number of new genes, including mammalian RNases 11-13, which are close relatives of the recently identified RNases 9 and 10. Gene expression data imply male-reproductive functions for RNases 9-13, although their sequences suggest the lack of ribonucleolytic activities. In contrast to the presence of 13-20 functional genes in mammals, chicken has only 3 RNase genes, which are evolutionarily close to mammalian RNase 5, like other nonmammalian RNases. This and other evidence suggests that the RNase A superfamily originated from an RNase 5-like gene and expanded in mammals. Together with the fact that multiple lineages of the superfamily, including RNases 2, 3, 5, and 7, have antipathogenic activities, we suggest that the superfamily started off as a host-defense mechanism in vertebrates. Consistent with this hypothesis, all members of the superfamily exhibit high rates of amino acid substitution as is commonly observed in immunity genes.     

Mapping, Phylogenetic and Expression Analysis of the RNase (RNaseA) Locus in Cattle

The mammalian secreted ribonucleases (RNases) comprise a large family of structurally related proteins displaying considerable sequence variation, and have been used in evolutionary studies. RNase 1 (RNase A) has been assumed to play a role in digestion, while other members have been suggested to contribute to host defence. Using the recently assembled bovine genome sequence, we characterised the complete repertoire of genes present in the RNaseA family locus in cattle, and compared this with the equivalent locus in the human and mouse genomes. Several additions and corrections to the earlier analysis of the RNase locus in the mouse genome are presented. The bovine locus encodes 19 RNases, of which only six have unambiguous equivalent genes in the other two species. Chromosomal mapping and phylogenetic analysis indicate that a number of distinct gene duplication events have occurred in the cattle lineage since divergence from the human and mouse lineages. Substitution analysis suggests that some of these duplicated genes are under evolutionary pressure for purifying selection and may therefore be important to the physiology of cattle. Expression analysis revealed that individual RNases have a wide pattern of expression, including diverse mucosal epithelia and immune-related cells and tissues. These data clarify the full repertoire of bovine RNases and their relationships to those in humans and mice. They also suggest that RNase gene duplication within the bovine lineage accompanied by altered tissue-specific expression has contributed a survival advantage.     

Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T₂ ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.     

Molecular variation at the self-incompatibility locus in natural populations of the genera Antirrhinum and Misopates

The self-incompatibility system of flowering plants is a classic example of extreme allelic polymorphism maintained by frequency-dependent selection. We used primers designed from three published Antirrhinum hispanicum S-allele sequences in PCR reactions with genomic DNA of plants sampled from natural populations of Antirrhinum and Misopates species. Not surprisingly, given the polymorphism of S-alleles, only a minority of individuals yielded PCR products of the expected size. These yielded 35 genomic sequences, of nine different sequence types of which eight are highly similar to the A. hispanicum S-allele sequences, and one to a very similar unpublished Antirrhinum S-like RNase sequence. The sequence types are well separated from the S-RNase sequences from Solanaceae and Rosaceae, and also from most known "S-like" RNase sequences (which encode proteins not involved in self-incompatibility). An association with incompatibility types has so far been established for only one of the putative S-alleles, but we describe evidence that the other sequences are also S-alleles. Variability in these sequences follows the pattern of conserved and hypervariable regions seen in other S-RNases, but no regions have higher replacement than silent diversity, unlike the results in some other species.     

A relic S-RNase is expressed in the styles of self-compatible Nicotiana sylvestris

We surveyed ribonuclease activity in the styles of Nicotiana spp. and found little or no activity in self-compatible species and in a self-compatible accession of a self-incompatible species. All self-incompatible species had high levels of ribonuclease activity in their style. Interestingly, one self-compatible species, N. sylvestris, had a level of stylar ribonuclease activity comparable to that of some self-incompatible Nicotiana species. A ribonuclease with biochemical properties similar to those of the self-incompatibility (S-)RNases of N. alata was purified from N. sylvestris styles. The N-terminal sequence of this protein was used to confirm the identity of a cDNA corresponding to the stylar RNase. The amino acid sequence deduced from the cDNA was related to those of the S-RNases and included the five conserved regions characteristic of these proteins. It appears that the N. sylvestris RNase may have evolved from the S-RNases and is an example of a 'relic S-RNase'. A number of features distinguish the N. sylvestris RNase from the S-RNases, and the role these may have played in the presumed loss of the self-incompatibility response during the evolution of this species are discussed.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.