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.     

丛枝菌根真菌对不同番茄品种抗根结线虫病的影响

【背景】根结线虫病害严重制约我国设施蔬菜的生产。丛枝菌根真菌(Arbuscular Mycorrhizal Fungi,AMF)作为土壤中最重要的有益真菌之一,可以促进植物生长,提高植物抗病性,减轻土传真菌和线虫病害的发生。在蔬菜保护地栽培中,AMF对于植物线虫病防治作用的研究受到了广泛关注。【目的】针对番茄生产中危害最严重的南方根结线虫(Meliodogyne incognita)病害问题,研究AMF和番茄品种不同组合的抗线虫效应,以期为菌根真菌作为生物防治剂和生物菌肥应用于实际生产提供技术基础。【方法】在灭菌土壤中,人工接种根结线虫,比较不同菌种Rhizophagus intraradices(Ri)、Acaulosporamellea(Am)及菌种组合Rhizophagusintraradices+Acaulosporamellea(Ri+Am)在不同番茄品种(感病品种蒙特卡罗和抗线虫品种仙客1号)上对南方根结线虫侵染和繁殖的影响,研究AMF对根结线虫的拮抗效应。另外,采用南方根结线虫连作发病的土壤,在感病品种蒙特卡洛上接种AMF混合菌种Ri+Am,番茄苗移栽入连作土壤中,测定各生长指标和调查根结和卵块数量,评价接种AMF处理对根结线虫病的防治效果。【结果】在灭菌土壤中,普通番茄品种蒙特卡罗的菌根效应显著优于抗线虫番茄品种仙客1号,表现为前者单位根重的根结和卵块的数量均比对照显著降低,而后者仅降低了卵块数量;蒙特卡罗上接种Ri+Am混合菌种的效果优于接种单一菌种Am和Ri;而仙客1号上接种Ri的效果更好。接种线虫也显著影响了AMF的侵染,而且对抗性品种仙客1号的影响更为明显。但除了接种Am的处理,大多数处理收获时菌根侵染率仍维持较高的水平(70%以上)。在连作土壤中,感病品种蒙特卡罗接种混合菌种Ri+Am具有较好的抗/耐线虫效应,主要表现为促进植株生长,显著降低根结和卵块数量,但菌根侵染率较灭菌土壤低,约为40%。【结论】综合以上结果,表明菌根化苗能够在一定程度上减轻根结线虫病的危害。土壤灭菌条件下,在感病和抗线虫番茄品种上接种AMF能够减轻线虫的侵染和繁殖,而且在感病品种上的防治效果更加显著。在连作土壤中,在番茄感病品种上接种AMF也表现较好的抗线虫效果。     

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/     

The map-1 gene family in root-knot nematodes, Meloidogyne spp.: a set of taxonomically restricted genes specific to clonal species

Taxonomically restricted genes (TRGs), i.e., genes that are restricted to a limited subset of phylogenetically related organisms, may be important in adaptation. In parasitic organisms, TRG-encoded proteins are possible determinants of the specificity of host-parasite interactions. In the root-knot nematode (RKN) Meloidogyne incognita, the map-1 gene family encodes expansin-like proteins that are secreted into plant tissues during parasitism, thought to act as effectors to promote successful root infection. MAP-1 proteins exhibit a modular architecture, with variable number and arrangement of 58 and 13-aa domains in their central part. Here, we address the evolutionary origins of this gene family using a combination of bioinformatics and molecular biology approaches. Map-1 genes were solely identified in one single member of the phylum Nematoda, i.e., the genus Meloidogyne, and not detected in any other nematode, thus indicating that the map-1 gene family is indeed a TRG family. A phylogenetic analysis of the distribution of map-1 genes in RKNs further showed that these genes are specifically present in species that reproduce by mitotic parthenogenesis, with the exception of M. floridensis, and could not be detected in RKNs reproducing by either meiotic parthenogenesis or amphimixis. These results highlight the divergence between mitotic and meiotic RKN species as a critical transition in the evolutionary history of these parasites. Analysis of the sequence conservation and organization of repeated domains in map-1 genes suggests that gene duplication(s) together with domain loss/duplication have contributed to the evolution of the map-1 family, and that some strong selection mechanism may be acting upon these genes to maintain their functional role(s) in the specificity of the plant-RKN interactions.     

Maize 9-lipoxygenase ZmLOX3 controls development, root-specific expression of defense genes, and resistance to root-knot nematodes

Root-knot nematodes (RKN) are severe pests of maize. Although lipoxygenase (LOX) pathways and their oxylipin products have been implicated in plant-nematode interactions, prior to this report there was no conclusive genetic evidence for the function of any plant LOX gene in such interactions. We showed that expression of a maize 9-LOX gene, ZmLOX3, increased steadily and peaked at 7 days after inoculation with Meloidogyne incognita RKN. Mu-insertional lox3-4 mutants displayed increased attractiveness to RKN and an increased number of juveniles and eggs. A set of jasmonic acid (JA)- and ethylene (ET)-responsive and biosynthetic genes as well as salicylic acid (SA)-dependent genes were overexpressed specifically in the roots of lox3-4 mutants. Consistent with this, levels of JA, SA, and ET were elevated in lox3-4 mutant roots, but not in leaves. Unlike wild types, in lox3-4 mutant roots, a phenylalanine ammonia lyase (PAL) gene was not RKN-inducible, suggesting a role for PAL-mediated metabolism in nematode resistance. In addition to these alterations in the defense status of roots, lox3-4 knockout mutants displayed precocious senescence and reduced root length and plant height compared with the wild type, suggesting that ZmLOX3 is required for normal plant development. Taken together, our data indicate that the ZmLOX3-mediated pathway may act as a root-specific suppressor of all three major defense signaling pathways to channel plant energy into growth processes, but is required for normal levels of resistance against nematodes.     

Arabidopsis thaliana genes expressed in the early compatible interaction with root-knot nematodes

In the compatible interaction between Arabidopsis thaliana and the endoparasitic nematode Meloidogyne incognita, galls are formed on the roots of the host plant. Differential display was used to identify alterations of gene expression in young A. thaliana root galls caused by M. incognita. Six genes were confirmed as plant genes by DNA gel blot hybridizations. Significant homology was found with a trypsin inhibitor, peroxidase, mitochondrial uncoupling protein, endomembrane protein, 20S proteasome alpha-subunit, and diaminopimelate decarboxylase. The cellular and temporal expression of each of the six genes was analyzed by mRNA in situ hybridizations.     

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.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum?×?almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.     

A molecular study of root-knot nematode-induced feeding sites

In a compatible interaction, root-knot nematodes ( Meloidogyne ) induce a sophisticated feeding site shortly after they have penetrated the plant root. The feeding site contains metabolically highly active giant cells. To gain insight into the molecular aspects that are typical for giant cells, a cDNA library from tomato roots infected with Meloidogyne incognita was differentially screened to find induced genes. Among the genes identified, two extensin genes ( Lemmi8 and Lemmi11 ) and a Lea -like gene ( Lemmi9 ) were studied further.     

Peroxiredoxins from the plant parasitic root-knot nematode, Meloidogyne incognita, are required for successful development within the host

Root-knot nematodes, Meloidogyne spp., are sedentary biotrophic parasites which are able to infest > 2000 plant species. After root invasion they settle sedentarily inside the vascular cylinder and maintain a compatible interaction for up to 8 weeks. Plant cells respond to pathogen attacks by producing reactive oxygen species (ROS). These ROS, in particular hydroperoxides, are important regulators of host-parasite interactions and partly govern the success or failure of disease. ROS producing and ROS scavenging enzymes from both the pathogen and the host finely tune the redox state at the host-pathogen interface. We have analysed the gene structure and organization of peroxiredoxins (prx) in Meloidogyne incognita and analysed their role in the establishment of the nematode in its host. Meloidogyne incognita has seven prx genes that can be grouped with other nematode prx into three clades. Clade B prx genes are more actively transcribed in parasitic stages compared with free-living pre-parasitic juveniles. We confirmed in vitro the activity of one of these, Mi-prx2.1, on hydrogen peroxide and butylhydroperoxide. We showed by ultrastructural immunocytochemistry the expression of clade B PRX proteins in the hypodermis and pseudocoelum beneath the tissues directly in contact with the environment, both in free-living and parasitic stages. Finally, knock-down of clade B prx genes led to a significant reduction in the ability of the nematodes to complete their life cycle in the host. The expression of clade B PRX proteins in the tissues in close contact with plant cells during parasitism and the impaired development of nematodes inside the host after clade B prx knock-down suggest an important role for these genes during infection.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.