Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes

Root-knot nematodes (RKN) of the genus Meloidogyne are biotrophic plant parasites of major agricultural importance, which exhibit very variable modes of reproduction, from classical amphimixis to mitotic parthenogenesis. This review focuses on those RKN species that reproduce exclusively by mitotic parthenogenesis (apomixis), in contrast to those that have meiotic/amphimitic events in their life cycle. Although populations of clonal organisms are often represented as being ecologically isolated and evolutionary inert, a considerable volume of literature provides evidence that asexual RKN are neither: they are widely distributed, extremely polyphagous, and amenable to selection and adaptive variation. The ancestors of the genus are unknown, but it is assumed that the parthenogenetic RKN have evolved from amphimictic species through hybridization and subsequent aneuploidization and polyploidization events. Molecular studies have indeed confirmed that the phylogenetic divergence between meiotic and mitotic RKN lineages occurred early, and have revealed an unexpected level of clonal diversity among populations within apomictic species. Laboratory experiments have shown that asexual RKN can rapidly adapt to new environmental constraints (eg host resistance), although with some fitness costs. Lastly, the molecular and chromosomal mechanisms that could contribute to genome plasticity leading to persistent genetic variation and adaptive evolution in apomictic RKN are discussed. It is concluded that RKN provide an excellent model system in which to study the dynamic nature and adaptive potential of clonal genomes.     

Modular architecture and evolution of the <Emphasis Type="Italic">map-1</Emphasis> gene family in the root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

In eukaryotes, repeat proteins (i.e. proteins that contain a tandem arrangement of repeated structural elements) are often considered as an extra source of variability, and gains and losses of repeats may be an important force driving the evolution and diversification of such proteins, that could allow fast adaptation to new environments. Here, we report genomic sequences of the MAP-1 protein family from of the asexual, plant-parasitic nematode Meloidogyne incognita. The encoded proteins exhibited highly conserved repeats of 13 and 58 aa, and variation in the number and arrangement of these repeats in the MAP-1 proteins was correlated with nematode (a)virulence, suggesting a possible role in the specificity of the plant–nematode interaction. Search in the complete genome sequence of M. incognita confirmed that a small gene family encoding proteins harboring conserved 58 and 13 aa-repeats is present in this nematode, and that the repetitive region of these proteins is modular. Both gene duplication and intragenic gain and loss of repeats have contributed to the complex evolutionary history of the map-1 gene family, and active selection pressure of the plant host probably induced recent additional gene loss, finally resulting in the present-day gene and repeat diversity observed among nematode lines. The genomic differences characterized here between avirulent and virulent individuals are assumed to reflect, at the DNA level, the adaptive capacity of these asexual root-knot nematodes.     

RKN Lethal DB: A database for the identification of Root Knot Nematode (Meloidogyne spp.) candidate lethal genes

Root Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating parasites that infect the roots of hundreds of plant species. RKN cannot live independently from their hosts and are the biggest contributors to the loss of the world''s primary foods. RNAi gene silencing studies have demonstrated that there are fewer galls and galls are smaller when RNAi constructs targeted to silence certain RKN genes are expressed in plant roots. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne Arenaria, Meloidogyne Chitwoodi, Meloidogyne Hapla, Meloidogyne Incognita, Meloidogyne Javanica, and Meloidogyne Paranaensis to that of the free living nematode Caenorhabditis elegans, to identify candidate genes that will be lethal to RKN when silenced or mutated. Our analysis yielded a number of such candidate lethal genes in RKN, some of which have been tested and proven to be effective in soybean roots. A web based database was built to house and allow scientists to search the data. This database will be useful to scientists seeking to identify candidate genes as targets for gene silencing to confer resistance in plants to RKN.

Availability

The database can be accessed from http://bioinformatics.towson.edu/RKN/     

High conservation of the differentially amplified MPA2 satellite DNA family in parthenogenetic root-knot nematodes

Sequence variability and distribution of a newly characterized MPA2 satellite DNA family are described in five root-knot nematode species of the genus Meloidogyne, the mitotic parthenogens M. paranaensis, M. incognita, M. arenaria and M. javanica, and the meiotic/mitotic M. hapla (isolates A and B, respectively). The lack of distinctive mutations and the considerable contribution (40.8%) of ancestral changes disclose an ancient satellite DNA which existed in the common ancestor of extant parthenogenetic species in the same or similar form and remained preserved for a period of at least 43 My. Nonuniformly distributed polymorphic sites along the satellite monomer suggest differences in constraints acting on particular sequence segments. Sequence diversity is clearly unaffected by significant differences in genomic abundance of the MPA2 satellite DNA in the examined species. Observed results suggest that the dynamics of this satellite DNA family might be in the first instance a consequence of characteristics of its nucleotide sequence and possible constraints imposed on it. Under conditions of mitotic and meiotic parthenogenesis, slow accumulation of mutations and slow replacement of old MPA2 sequence variants with new ones may be equivalent to the dynamics of some satellite DNA sequences conserved for extremely long evolutionary periods in sexual species.     

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.     

Comparative proteomic analysis of the response of fibrous roots of nematode-resistant and -sensitive sweet potato cultivars to root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

As a major root-knot nematode (RKN), Meloidogyne incognita causes serious losses in the yield of sweet potato (Ipomoea batatas L.). To successfully colonize the host plant, RKNs elicit changes of dramatic physiological and morphological features in the plants. The expression of several genes is regulated as the nematode establishes its feeding site. Therefore, in this study, we analyzed the proteomes in the fibrous roots of sweet potato plants by an infection of RKN to understand the effect of the infection on the plant root regions. This study revealed differences in proteomes of the RKN-resistant sweet potato cultivar Juhwangmi and RKN-sensitive cultivar Yulmi. During plant growth, Juhwangmi plants were shown to be more resistant to M. incognita than Yulmi plants. No M. incognita egg formation was observed in Juhwangmi plants, whereas 587 egg masses were formed in Yulmi plants. Differentially expressed 64 spots were confirmed by proteomic analysis using 2-D gel electrophoresis with three spots up-regulated in the two cultivars during RKN infection. Of these 64 protein spots, 20 were identified as belonging to such different functional categories as the defense response, cell structure, and energy metabolism. This study provides insight into the molecular and biochemical mechanics of the defense response and metabolism of sweet potato plant during nematode invasion. We anticipate that this study will also provide a molecular basis for useful crop breeding and the development of nematode-tolerant plants.     

Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication events in the DUF606 family

X-ray crystallography has revealed that many integral membrane proteins consist of two domains with a similar fold but opposite (antiparallel) orientation in the membrane. The proteins are believed to have evolved by gene duplication and gene fusion events from a dual topology ancestral membrane protein, that adapted both orientations in the membrane and formed antiparallel homodimers. Here, we present a detailed analysis of the DUF606 family of bacterial membrane proteins that contains the entire collection of intermediate states of such an evolutionary pathway: single genes that would code for dual topology homodimeric proteins, paired genes coding for homologous proteins with a fixed but opposite orientation in the membrane that would form heterodimers, and fused genes that encode antiparallel two-domain fusion proteins. Two types of paired genes can be discriminated corresponding to the order in which the genes coding for the two oppositely oriented proteins occur in the operon. On the protein level, the heterodimers resulting from the two types of gene pairs are indistinguishable. In contrast, two types of fused genes corresponding to the two possible orders in which the oppositely oriented domains are present in the encoded proteins, do result in discernible types of proteins. The large number of genetic and protein states in the DUF606 family allowed for a detailed phylogenic analysis that revealed a total of nine independent duplication events in the DUF606 family, five of which resulted in paired genes, and four resulted in fused genes. Noticeably, there was no evidence for a sequential mechanism in which fusions evolve from a pair of genes. Rather, an evolutionary mechanism is proposed by which antiparallel two-domain proteins are the direct result of a gene duplication event. Combining the phylogeny of proteins and hosting microorganisms allowed for a reconstruction of the evolutionary pathway.     

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.     

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2–13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.     

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.     

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins

Accumulation of the non-reducing disaccharide trehalose is associated with desiccation tolerance during anhydrobiosis in a number of invertebrates, but there is little information on trehalose biosynthetic genes in these organisms. We have identified two trehalose-6-phosphate synthase (tps) genes in the anhydrobiotic nematode Aphelenchus avenae and determined full length cDNA sequences for both; for comparison, full length tps cDNAs from the model nematode, Caenorhabditis elegans, have also been obtained. The A. avenae genes encode very similar proteins containing the catalytic domain characteristic of the GT-20 family of glycosyltransferases and are most similar to tps-2 of C. elegans; no evidence was found for a gene in A. avenae corresponding to Ce-tps-1. Analysis of A. avenae tps cDNAs revealed several features of interest, including alternative trans-splicing of spliced leader sequences in Aav-tps-1, and four different, novel SL1-related trans-spliced leaders, which were different to the canonical SL1 sequence found in all other nematodes studied. The latter observation suggests that A. avenae does not comply with the strict evolutionary conservation of SL1 sequences observed in other species. Unusual features were also noted in predicted nematode TPS proteins, which distinguish them from homologues in other higher eukaryotes (plants and insects) and in micro-organisms. Phylogenetic analysis confirmed their membership of the GT-20 glycosyltransferase family, but indicated an accelerated rate of molecular evolution. Furthermore, nematode TPS proteins possess N- and C-terminal domains, which are unrelated to those of other eukaryotes: nematode C-terminal domains, for example, do not contain trehalose-6-phosphate phosphatase-like sequences, as seen in plant and insect homologues. During onset of anhydrobiosis, both tps genes in A. avenae are upregulated, but exposure to cold or increased osmolarity also results in gene induction, although to a lesser extent. Trehalose seems likely therefore to play a role in a number of stress responses in nematodes.     

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T₁, T₂ and T₃ transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

     

Plant resistance genes: molecular and genetic organization,function and evolution

Remarkable progress is achieved now in comprehension of mechanisms that determine functioning of genes responsible for plants' phytopathogenic resistance (genes R). Cloning of great number of Monocotyledones and Dicotyledones resistance genes show that most of proteins coded by these genes have conserved amino-acid motives, which show high homology to amino-acid motives of proteins with well-designated function. Common structures for most proteins produced by genes R include nucleotide-blinding site (NBS), leucine-rich repeat (LRR), site containing homology with the cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor (TIR), coiled-coil structure (CC), transmembrane domain (TM), and serine/threonine proteinkinase domain (PK). They are combined within the basic classes of resistance genes proteins as follows: TIR-NBS-LRR, CC-NBS-LLRR, NBS-LRR, PK, TM-CC, LRR-TM, LRR-TM-PK. The domains of proteins produced by plant resistance genes cause specific recognition of avirulence genes products and activate signaling cascade, which gives rise to resistance reaction. Some classes of plant resistance genes probably have the same evolutionary origin as the genes that control the innate immunity of ancient animals. The evolution of plant R genes proceeds primarily by duplication and equal or unequal meiotic recombination. The research on genes R functioning besides its theoretical value is a matter of considerable practical interest for construction of plant genotypes resistant against harmful organisms. The progress in comprehension of mechanisms responsible for specificity of avirulence determinants in phytopathogenic organisms recognition makes possible the creation of artificial resistance genes.     

Evolutionary history of tissue kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.     

Meiotic parthenogenesis in a root-knot nematode results in rapid genomic homozygosity

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F(2) lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (<3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.