Uninfected Roots Research Articles

Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis

The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant–fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.

Secondary metabolites related to the resistance of Psidium spp. against the nematode Meloidogyneenterolobii

The guava tree (Psidium guajava) is a tropical species native to South America and is recognized as the 11th most economically important fruit tree in Brazil. However, the presence of the nematode Meloidogyne enterolobii and the fungus Fusarium solani in the roots of guava plants leads to the development of root galls, causing significant damage. In contrast, the species P. guineense and P. cattleianum have been identified as resistant and immune to the nematode, respectively. In this study, the researchers aimed to compare the metabolomic profiles of infected and uninfected roots of P. guajava, P. cattleianum, and P. guineense using mass spectrometry coupled with liquid chromatography (LC-MS). The goal was to identify secondary metabolites that could potentially be utilized as biochemical resources for nematode control. The findings of the study demonstrated that the plant metabolism of all three species undergoes alterations in response to the phytopathogen inoculation. By employing molecular networks, the researchers identified that the secondary metabolites affected by the infection, whether produced or suppressed, are primarily of a polar chemical nature. Further analysis of the database confirmed the polar nature of the regulated substances after infection, specifically hydrolysable tannins and lignans in P. guineense and P. cattleianum. Interestingly, a group of non-polar substances belonging to the terpene class was also identified in the resistant and immune species. This suggests that these terpenes may act as inhibitors of M. enterolobii, working as repellents or as molecules that can reduce oxidative stress during the infection process, thus enhancing the guava resistance to the nematode. Overall, this study provides valuable insights into the metabolic alterations occurring in different Psidium spp. in response to M. enterolobii infection. The identification of specific secondary metabolites, particularly terpenes, opens up new possibilities for developing effective strategies to control the nematode and enhance guava resistance.

Microbial landscapes of the rhizosphere soils and roots of Luffa cylindrica plant associated with Meloidogyne incognita.

The root-knot nematodes (RKN), especially Meloidogyne spp., are globally emerging harmful animals for many agricultural crops. To explore microbial agents for biological control of these nematodes, the microbial communities of the rhizosphere soils and roots of sponge gourd (Luffa cylindrica) infected and non-infected by M. incognita nematodes, were investigated using culture-dependent and -independent methods. Thirty-two culturable bacterial and eight fungal species, along with 10,561 bacterial and 2,427 fungal operational taxonomic units (OTUs), were identified. Nine culturable bacterial species, 955 bacterial and 701 fungal OTUs were shared in both four groups. More culturable bacterial and fungal isolates were detected from the uninfected soils and roots than from the infected soils and roots (except no fungi detected from the uninfected roots), and among all samples, nine bacterial species (Arthrobacter sp., Bacillus sp., Burkholderia ambifaria, Enterobacteriaceae sp., Fictibacillus barbaricus, Microbacterium sp., Micrococcaceae sp., Rhizobiaceae sp., and Serratia sp.) were shared, with Arthrobacter sp. and Bacillus sp. being dominant. Pseudomonas nitroreducens was exclusively present in the infested soils, while Mammaliicoccus sciuri, Microbacterium azadirachtae, and Priestia sp., together with Mucor irregularis, Penicillium sp., P. commune, and Sordariomycetes sp. were found only in the uninfected soils. Cupriavidus metallidurans, Gordonia sp., Streptomyces viridobrunneus, and Terribacillus sp. were only in the uninfected roots while Aspergillus sp. only in infected roots. After M. incognita infestation, 319 bacterial OTUs (such as Chryseobacterium) and 171 fungal OTUs (such as Spizellomyces) were increased in rhizosphere soils, while 181 bacterial OTUs (such as Pasteuria) and 166 fungal OTUs (such as Exophiala) rose their abundance in plant roots. Meanwhile, much more decreased bacterial or fungal OTUs were identified from rhizosphere soils rather than from plant roots, exhibiting the protective effects of host plant on endophytes. Among the detected bacterial isolates, Streptomyces sp. TR27 was discovered to exhibit nematocidal activity, and B. amyloliquefaciens, Bacillus sp. P35, and M. azadirachtae to show repellent potentials for the second stage M. incognita juveniles, which can be used to develop RKN bio-control agents. These findings provided insights into the interactions among root-knot nematodes, host plants, and microorganisms, which will inspire explorations of novel nematicides.

Sucrose-induced auxin conjugate hydrolase restores symbiosis in a Medicago cytokinin perception mutant.

Rhizobia-legume interactions recruit cytokinin for the induction of nodule primordia in the cortex. Cytokinin signaling regulates auxin transport and biosynthesis, causing local auxin accumulation, which triggers cortical cell division. Since sugar signaling can trigger auxin responses, we explored whether sugar treatments could rescue symbiosis in the Medicago truncatula cytokinin response 1 (cre1) mutant. Herein, we demonstrate that sucrose and its nonmetabolizable isomer turanose can trigger auxin response and recover functional symbiosis in cre1, indicating sucrose signaling to be necessary for the restoration of symbiosis. In both M. truncatula A17 (wild type) and cre1, sucrose signaling significantly upregulated IAA-Ala Resistant 3 (IAR33), encoding an auxin conjugate hydrolase, in rhizobia-infected as well as in uninfected roots. Knockdown of IAR33 (IAR33-KD) significantly reduced nodulation in A17, highlighting the importance of deconjugation-mediated auxin accumulation during nodule inception. In cre1, IAR33-KD restricted the sucrose-mediated restoration of functional symbiosis, suggesting that deconjugation-mediated auxin accumulation plays a key role in the absence of CRE1-mediated auxin biosynthesis and transport control. Overexpression of IAR33 also restored functional symbiosis in cre1, further suggesting that IAR33 mediates auxin accumulation in response to sucrose signaling. Since all the observed sucrose-mediated responses were common to A17 and cre1, deconjugation-mediated auxin response appeared to be independent of CRE1, which normally governs local auxin accumulation in the presence of rhizobia. We propose that sucrose-dependent restoration of symbiosis in cre1 occurs by the activation of IAR33-mediated auxin deconjugation.

Comparative Root Morpho-Anatomical Studies On Meloidogyne Graminicola Resistant And Susceptible Rice Genotypes

The work characterizes root morpho-anatomical changes in rice due to infestation of rice root-knot nematode, Meloidogyne graminicola. The susceptible rice genotype, Satabdi (IET-4786) and resistant rice genotypes Marichsal and Lakshmansal were inoculated with three different geographic isolates of M. graminicola viz., Mg1 (Ashui, West Medinipur), Mg2 (Sumitrapur, West Medinipur) and Mg3 (Gayeshpur, Nadia). The results of demonstrated that, transverse section of morpho-anatomical structure of Mg1 isolate inoculated root at 25 DAI to susceptible and resistant rice genotypes viz., Satabdi and Marichsal showed presence of adult females and egg-mass in the cortex which symbolized a successful host parasitic relationship. Organization of cellular architecture of adventitious root of rice genotypes Satabdi and Marichsal when inoculated with Mg2 isolate then nematode infestation led to drastic changes as evidenced by the disorganization of cortical cells along with dissolution of epidermal, exodermal and sclerenchymatous cells at 10 DAI. The rice genotype, Lakshmansal did not reveal any changes in the root morpho-anatomy when inoculated with Mg2 isolate at 10 DAI. The cross section of the roots of Satabdi and Marichsal revealed complete obliteration of root anatomical structures along with formation of multinucleate giant cells, cavities in the stele owing to nematode injury along with females and egg-masses at 10 and 25 DAI of Mg3 isolate. The rice genotype Lakshmansal, was characterized as resistant against all three isolates of Mg as confirmed by histological study of roots. Cortical thickness of uninfected rice roots was comparatively lesser than infected ones, while maximum stele diameter was recorded with Marichsal when inoculated with Mg1 isolate i.e. 523.01 µm at 25 DAI. Outer portion of the root was also less than infected ones. Giant cell diameter of Satabdi and Marichsal ranged from 43.45-107.46 µm and 30.68-102.67 µm, respectively, indicating successful establishment of host-nematode interaction.

The DNA methylation landscape of the root-knot nematode-induced pseudo-organ, the gall, in Arabidopsis, is dynamic, contrasting over time, and critically important for successful parasitism.

Root-knot nematodes (RKNs) induce giant cells (GCs) within galls which are characterized by large-scale gene repression at early stages. However, the epigenetic mechanism(s) underlying gene silencing is (are) still poorly characterized. DNA methylation in Arabidopsis galls induced by Meloidogyne javanica was studied at crucial infection stages (3 d post-infection (dpi) and 14 dpi) using enzymatic, cytological, and sequencing approaches. DNA methyltransferase mutants (met1, cmt2, cmt3, cmt2/3, drm1/2, ddc) and a DNA demethylase mutant (ros1), were analyzed for RKN resistance/tolerance, and galls were characterized by confocal microscopy and RNA-seq. Early galls were hypermethylated, and the GCs were found to be the major contributors to this hypermethylation, consistent with the very high degree of gene repression they exhibit. By contrast, medium/late galls showed no global increase in DNA methylation compared to uninfected roots, but exhibited large-scale redistribution of differentially methylated regions (DMRs). In line with these findings, it was also shown that DNA methylation and demethylation mutants showed impaired nematode reproduction and gall/GC-development. Moreover, siRNAs that were exclusively present in early galls accumulated at hypermethylated DMRs, overlapping mostly with retrotransposons in the CHG/CG contexts that might be involved in their repression, contributing to their stability/genome integrity. Promoter/gene methylation correlated with differentially expressed genes encoding proteins with basic cell functions. Both mechanisms are consistent with reprogramming host tissues for gall/GC formation. In conclusion, RNA-directed DNA methylation (RdDM; DRM2/1) pathways, maintenance methyltransferases (MET1/CMT3) and demethylation (ROS1) appear to be prominent mechanisms driving a dynamic regulation of the epigenetic landscape during RKN infection.

Nematicidal rhizobacteria with plant growth-promoting traits associated with tomato in root-knot infested polyhouses

BackgroundIn polyhouse cultivation of tomato (Solanum lycopersicum) in root-knot (Meloidogyne incognita) infested soils, several accessions remained free of nematode infection. It was hypothesized that such plants that were free of root-knot infection in the selected polyhouses were possibly harboring potent nematode antagonistic microbes. Therefore, attempts were made to isolate root-associated rhizobacteria from nematode infected and uninfected roots of 7 tomato accessions. Eighty morphologically distinct isolates were multiplied in nutrient broth for 48 h to test the presence of nematotoxic metabolites. Laboratory bioassays were conducted using their cell-free filtrates to observe mortality in M. incognita juveniles (J2s) at 24 h. The most potent ones were identified through molecular characterization (16S rDNA) and assayed for plant growth promotion traits, seed germination and seedling vigor and bioefficacy against M. incognita infecting polyhouse tomato.ResultsRhizobacterial densities were estimated from the root samples using 3 media. In all the media, the average numbers of colony forming units (cfu) from uninfected roots were significantly higher than the infected roots. The maximum numbers of colonies were observed in soil plate count agar. The extracellular metabolites caused juvenile mortality in the range of 60.67–100% from isolates associated with nematode uninfected roots of accessions H308, H299 and H266, as compared to isolates from nematode infected roots of accessions H195, BSS99, H178/F4 and H88, which caused mortality in the range of 52.33 to 73.67%. Four isolates (B9, B16, B26 and B31) caused 100% J2 mortality, followed by 13 isolates (B7, B8, B13, B14, B17, B18, B21, B22, B25, B28, B29, B32 and B33) that caused 90–95%, 12 isolates caused 80–85% and 51 isolates caused < 85% J2 mortality, when exposed to bacterial metabolites in cell-free filtrates for 24 h. The isolates B9, B16, B26 and B31 were identified based on 16S rRNA sequence analysis as Bacillus pumilus, B. megaterium, B. subtilis and B. cereus, respectively. These 4 bacterial isolates possessed plant growth promotion traits like production of IAA, ammonia, catalase and chitinase with the ability to solubilize zinc and phosphate. These enhanced tomato seed germination and seedling vigor, and their application in soil resulted in significant increase in root and shoot length of tomato seedlings.ConclusionsIn the present investigation, the 21% bacterial colonies (17 isolates) that caused nemato toxicity in the range of 90 to 100% and another 15% (12 isolates) that caused juvenile mortality in the range of 80 to 90% were from uninfected tomato accessions, indicating their role in protecting the plants against root-knot nematode (RKN) infection. The 4 bacterial isolates characterized from uninfected plants possessed high nematicidal potential with plant growth promotion (PGP) traits that enhanced tomato seed germination and seedling vigor index. The isolates can be utilized for root-knot nematode pest management in polyhouse cultivation.

From Raffinose Family Oligosaccharides to Sucrose and Hexoses: Gene Expression Profiles Underlying Host-to-Nematode Carbon Delivery in Cucumis sativus Roots.

Root-knot nematodes (Meloidogyne incognita) induce specific feeding sites in cucumber roots where they absorb carbon nutrients from the host, thereby turning the feeding sites into a strong sink for assimilates. Nematode infection may alter host sugar metabolism in the roots of sucrose-transporting species. However, much less is known about the species translocating raffinose family oligosaccharides (RFOs), such as cucumber. To address this knowledge gap, the dynamics of RFOs and sucrose metabolisms, two major sugar-metabolism processes, in cucumber roots during nematode infection at transcription and protein levels were analyzed. In the nematode-infected root, the expressions of RFO-synthesis genes, CsRS (Raffinose Synthase) and CsGolS1 (Galactinol Synthase 1), were upregulated at early stage, but were significantly decreased, along with CsSTS (Stachyose Synthase), at the late stage during nematode infection. By contrast, α-galactosidase hydrolyzed RFOs into sucrose and galactose, whose encoding genes was suppressed (CsaGA2) at early stage and then elevated (CsaGA2, 4, and CsAGA1) at the late stage of nematode infection. Consistently, stachyose level was significantly increased by ∼2.5 times at the early stage but reduced at the late stage of infection in comparison with the uninfected roots, with a similar trend found for raffinose and galactinol. Moreover, the genes encoding sucrose synthase and cell wall invertase, which are responsible for sucrose degrading, were differentially expressed. In addition, sugar transporter, CsSUT4, was enhanced significantly after nematode infection at early stage but was suppressed at the late stage. Based on the observation and in connection with the information from literature, the RFOs play a role in the protection of roots during the initial stage of infection but could be used by nematode as C nutrients at the late stage.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Non-coding RNAs in the interaction between rice and Meloidogyne graminicola

BackgroundRoot knot nematodes (RKN) are plant parasitic nematodes causing major yield losses of widely consumed food crops such as rice (Oryza sativa). Because non-coding RNAs, including small interfering RNAs (siRNA), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are key regulators of various plant processes, elucidating their regulation during this interaction may lead to new strategies to improve crop protection. In this study, we aimed to identify and characterize rice siRNAs, miRNAs and lncRNAs responsive to early infection with RKN Meloidogyne graminicola (Mg), based on sequencing of small RNA, degradome and total RNA libraries from rice gall tissues compared with uninfected root tissues.ResultsWe found 425 lncRNAs, 3739 siRNAs and 16 miRNAs to be differentially expressed between both tissues, of which a subset was independently validated with RT-qPCR. Functional prediction of the lncRNAs indicates that a large part of their potential target genes code for serine/threonine protein kinases and transcription factors. Differentially expressed siRNAs have a predominant size of 24 nts, suggesting a role in DNA methylation. Differentially expressed miRNAs are generally downregulated and target transcription factors, which show reduced degradation according to the degradome data.ConclusionsTo our knowledge, this work is the first to focus on small and long non-coding RNAs in the interaction between rice and Mg, and provides an overview of rice non-coding RNAs with the potential to be used as a resource for the development of new crop protection strategies.

Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions.

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

Characterization and Genetic Mapping of Black Root Rot Resistance in Gossypium arboreum L.

Black root rot (BRR) is an economically important disease of cotton and other crops, especially in cooler regions with short growing seasons. Symptoms include black discoloration of the roots, reduced number of lateral roots and stunted or slow plant growth. The cultivated tetraploid Gossypium species are susceptible to BRR. Resistance to BRR was identified in G. arboreum accession BM13H and is associated with reduced and restricted hyphal growth and less sporulation. Transcriptome analysis indicates that BM13H responds to infection at early time points 2- and 3-days post-inoculation, but by day 5, few differentially expressed genes are observed between infected and uninfected roots. Inheritance of BM13H resistance to BRR was evaluated in an F6 recombinant inbred population and shows a single semi-dominant locus conferring resistance that was fine mapped to a region on chromosome 1, containing ten genes including five putative resistance-like genes.

Cyst Nematode Infection Elicits Alteration in the Level of Reactive Nitrogen Species, Protein S-Nitrosylation and Nitration, and Nitrosoglutathione Reductase in Arabidopsis thaliana Roots.

Reactive nitrogen species (RNS) are redox molecules important for plant defense against pathogens. The aim of the study was to determine whether the infection by the beet cyst nematode Heterodera schachtii disrupts RNS balance in Arabidopsis thaliana roots. For this purpose, measurements of nitric oxide (NO), peroxynitrite (ONOO−), protein S-nitrosylation and nitration, and nitrosoglutathione reductase (GSNOR) in A. thaliana roots from 1 day to 15 days post-inoculation (dpi) were performed. The cyst nematode infection caused generation of NO and ONOO− in the infected roots. These changes were accompanied by an expansion of S-nitrosylated and nitrated proteins. The enzyme activity of GSNOR was decreased at 3 and 15 dpi and increased at 7 dpi in infected roots, whereas the GSNOR1 transcript level was enhanced over the entire examination period. The protein content of GSNOR was increased in infected roots at 3 dpi and 7 dpi, but at 15 dpi, did not differ between uninfected and infected roots. The protein of GSNOR was detected in plastids, mitochondria, cytoplasm, as well as endoplasmic reticulum and cytoplasmic membranes. We postulate that RNS metabolism plays an important role in plant defense against the beet cyst nematode and helps the fine-tuning of the infected plants to stress sparked by phytoparasitic nematodes.

Modulation of Arabidopsis Flavonol Biosynthesis Genes by Cyst and Root-Knot Nematodes.

Although it is well established that flavonoid synthesis is induced in diverse plant species during nematode parasitism, little is known about the regulation of genes controlling flavonol biosynthesis during the plant–nematode interaction. In this study, expression of the Arabidopsis thaliana flavonol-specific transcription factor, AtMYB12, the flavonol synthase genes, AtFLS1, 2, 3, 4, and 5, and the gene encoding the central flavonoid enzyme, chalcone synthase (AtCHS), were examined in plant roots during infection by Heterodera schachtii (sugar beet cyst) and Meloidogyne incognita (root-knot) nematodes. These experiments showed that AtMYB12 was transiently upregulated at 9 dpi in syncytia associated with sugar beet cyst nematode infection and that an Atmyb12-deficient line was less susceptible to the parasite. This suggests that, rather than contributing to plant defense, this gene is essential for productive infection. However, the AtCHS and AtFLS1 genes, which are controlled by AtMYB12, did not exhibit a similar transient increase, but rather were expressly downregulated in syncytia relative to adjacent uninfected root tissue. Genetic analyses further indicated that AtFLS1 contributes to plant defense against Cyst nematode infection, while other AtFLS gene family members do not, consistent with prior reports that these other genes encode little or no enzyme activity. Together, these findings indicate a role of AtMyb12 in promoting the early stages of Cyst nematode infection, while flavonols produced through the action of AtFLS1 are essential for plant defense. On the other hand, a transient induction of AtMYB12 was not observed in galls produced during root-knot nematode infection, but this gene was instead substantially downregulated, starting at the 9 dpi sampling point, as were AtCHS and AtFLS1. In addition, both the AtMYB12- and AtFLS1-deficient lines were more susceptible to infection by this parasite. There was again little evidence for contributions from the other AtFLS gene family members, although an AtFLS5-deficient line appeared to be somewhat more susceptible to infection. Taken together, this study shows that sugar-beet cyst and root-knot nematodes modulate differently the genes involved in flavonol biosynthesis in order to successfully infect host roots and that AtFLS1 may be involved in the plant basal defense response against nematode infection.

Molecular Changes Concomitant with Vascular System Development in Mature Galls Induced by Root-Knot Nematodes in the Model Tree Host Populus tremula × P. alba.

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita, a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

Transcriptomic response in symptomless roots of clubroot infected kohlrabi (Brassica oleracea var. gongylodes) mirrors resistant plants

BackgroundClubroot disease caused by Plasmodiophora brassicae (Phytomyxea, Rhizaria) is one of the economically most important diseases of Brassica crops. The formation of hypertrophied roots accompanied by altered metabolism and hormone homeostasis is typical for infected plants. Not all roots of infected plants show the same phenotypic changes. While some roots remain uninfected, others develop galls of diverse size. The aim of this study was to analyse and compare the intra-plant heterogeneity of P. brassicae root galls and symptomless roots of the same host plants (Brassica oleracea var. gongylodes) collected from a commercial field in Austria using transcriptome analyses.ResultsTranscriptomes were markedly different between symptomless roots and gall tissue. Symptomless roots showed transcriptomic traits previously described for resistant plants. Genes involved in host cell wall synthesis and reinforcement were up-regulated in symptomless roots indicating elevated tolerance against P. brassicae. By contrast, genes involved in cell wall degradation and modification processes like expansion were up-regulated in root galls. Hormone metabolism differed between symptomless roots and galls. Brassinosteroid-synthesis was down-regulated in root galls, whereas jasmonic acid synthesis was down-regulated in symptomless roots. Cytokinin metabolism and signalling were up-regulated in symptomless roots with the exception of one CKX6 homolog, which was strongly down-regulated. Salicylic acid (SA) mediated defence response was up-regulated in symptomless roots, compared with root gall tissue. This is probably caused by a secreted benzoic acid/salicylic acid methyl transferase from the pathogen (PbBSMT), which was one of the highest expressed pathogen genes in gall tissue. The PbBSMT derived Methyl-SA potentially leads to increased pathogen tolerance in uninfected roots.ConclusionsInfected and uninfected roots of clubroot infected plants showed transcriptomic differences similar to those previously described between clubroot resistant and susceptible hosts. The here described intra-plant heterogeneity suggests, that for a better understanding of clubroot disease targeted, spatial analyses of clubroot infected plants will be vital in understanding this economically important disease.

Effect of Root-knot Nematode (Meloidogyne incognita) on the Nodulation of Some Varieties of Cowpea (Vigna unguiculata L. Walp)

Abstract Screenhouse and field trials were conducted at the University of Ilorin, Nigeria in 2013 and 2014 to investigate the effect of root knot nematode (Meloidogyne incognita) on the nodulation of five varieties of cowpea (IT89KD-288, IT82D-994, IT93K-452-1, T89K-391 and TT97 568-18). Half of the experimental plants were inoculated with Meloidogyne incognita eggs (n = 1500) while the remaining half served as uninoculated control plants. Data on growth and yield of cowpea, root gall, nodulation, and nematodes population densities in nodules were collected. Other parameters assessed included colour of dissected infected and uninfected nodules, texture of the nodules and varietal performance of the cowpeas. The results of both screenhouse and field trials were consistent in yield and visual observations of texture and colour. The yield of nematode-free varieties was significantly higher than that of M. incognita-infected ones. Infected nodules were dark brown in colour while the healthy ones were pink. For the field trials, nodulation counts in nematode inoculated plants were inversely proportional to the number of galls in their uninoculated counterparts in some varieties. Of all varieties, IT89KD-288 was most resistant to M. incognita. Due to high cost involved in the management of nematodes, especially with synthetic chemicals, variety IT89KD-288 is recommended for use in nematode endemic areas in Nigeria. Further study needs to be done on the histopathology of the infected and uninfected roots and nodules of these cowpea varieties.

Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass.

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5' terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita

BackgroundRoot-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways.ResultsSmall non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23–24 nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation.ConclusionsCorrelation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Silenced retrotransposons are major rasiRNAs targets in Arabidopsis galls induced by Meloidogyne javanica

Root-knot nematodes (RKNs, Meloidogyne spp.) are sedentary biotrophic pathogens that establish within the vascular cylinder of plant roots, forming a gall and inducing several feeding cells, giant cells (GCs), essential for completion of their life cycle. GCs suffer gene expression changes, repeated mitosis and endoreduplication events. Transcriptomics has revealed that an extensive down-regulation of transcripts, a molecular signature of early-developing galls and GCs that is conserved in tomato and Arabidopsis, may be achieved through small RNA (sRNA) gene silencing pathways. The role of some microRNAs (miRNAs) in plant-RKN interactions has recently been addressed, but little is known about the regulatory roles of other sRNA types. Here, we perform a differential accumulation analysis to show which repeat-associated small interfering RNAs (rasiRNAs) are distinctive or enriched in early Arabidopsis galls vs. uninfected roots. Those distinctive from galls are preferentially located in pericentromeric regions with predominant sizes of 24 and 22 nucleotides. Gall-distinctive rasiRNAs target primarily GYPSY and COPIA retrotransposons, which show a marked repression in galls vs. uninfected roots. Infection tests and phenotypic studies of galls from Meloidogyne javanica in Arabidopsis mutants impaired in post-transcriptional gene silencing and/or canonical RNA-directed DNA methylation (RdDM) pathways, as well as quantitative polymerase chain reaction analysis, suggest the implication of canonical and non-canonical RdDM pathways during gall formation, possibly through the regulation of retrotransposons. This process may be crucial for the maintenance of genome integrity during the reprogramming process of galls/GCs from their vascular precursor cells, and/or to ensure a faithful DNA replication during the repeated mitosis/endoreduplication that concurs with feeding site formation.