GmSABP2-1 encodes methyl salicylate esterase and functions in soybean defense against soybean cyst nematode.

KEY MESSAGE: The soybean gene GmSABP2-1 encodes methyl salicylate esterase and its overexpression led to significant reduction in development of pathogenic soybean cyst nematode. Soybean cyst nematode (SCN, Heterodera glycines) is one of the most devastating pests of soybean (Glycine max L. Merr.). In searching for SCN-defense genes, a soybean gene of the methylesterase (MES) family was found to be upregulated in an SCN-resistant soybean line and downregulated in an SCN-susceptible line upon SCN infection. This gene was designated as GmSABP2-1. Here, we report on biochemical and overexpression studies of GmSABP2-1 to examine its possible function in SCN resistance. The protein encoded by GmSABP2-1 is closely related to known methyl salicylate esterases. To determine the biochemical function of GmSABP2-1, a full-length cDNA of GmSABP2-1 was cloned into a protein expression vector and expressed in Escherichia coli. The resulting recombinant GmSABP2-1 was demonstrated to catalyze the demethylation of methyl salicylate. The biochemical properties of GmSABP2-1 were determined. Its apparent Km value was 46.2 ± 2.2 µM for methyl salicylate, comparable to those of the known methyl salicylate esterases. To explore the biological significance of GmSABP2-1 in soybean defense against SCN, we first overexpressed GmSABP2-1 in transgenic hairy roots of an SCN-susceptible soybean line. When infected with SCN, GmSABP2-1-overexpressing hairy roots showed 84.5% reduction in the development of SCN beyond J2 stage. To provide further genetic evidence for the role of GmSABP2-1 in SCN resistance, stable transgenic soybean plants overexpressing GmSABP2-1 were produced. Analysis of the GmSABP2-1-overexpressing lines showed a significant reduction in SCN development compared to non-transgenic plants. In conclusion, we demonstrated that GmSABP2-1 encodes methyl salicylate esterase and functions as a resistance-related gene against SCN.

The soybean immune receptor GmBIR1 regulates host transcriptome, spliceome, and immunity during cyst nematode infection.

BAK1-INTERACTING RECEPTOR LIKE KINASE1 (BIR1) is a negative regulator of various aspects of disease resistance and immune responses. Here, we investigated the functional role of soybean (Glycine max) BIR1 (GmBIR1) during soybean interaction with soybean cyst nematode (SCN, Heterodera glycines) and the molecular mechanism through which GmBIR1 regulates plant immunity. Overexpression of wild-type variant of GmBIR1 (WT-GmBIR1) using transgenic soybean hairy roots significantly increased soybean susceptibility to SCN, whereas overexpression of kinase-dead variant (KD-GmBIR1) significantly increased plant resistance. Transcriptome analysis revealed that genes oppositely regulated in WT-GmBIR1 and KD-GmBIR1 upon SCN infection were enriched primarily in defense and immunity-related functions. Quantitative phosphoproteomic analysis identified 208 proteins as putative substrates of the GmBIR1 signaling pathway, 114 of which were differentially phosphorylated upon SCN infection. In addition, the phosphoproteomic data pointed to a role of the GmBIR1 signaling pathway in regulating alternative pre-mRNA splicing. Genome-wide analysis of splicing events provided compelling evidence supporting a role of the GmBIR1 signaling pathway in establishing alternative splicing during SCN infection. Our results provide novel mechanistic insights into the function of the GmBIR1 signaling pathway in regulating soybean transcriptome and spliceome via differential phosphorylation of splicing factors and regulation of splicing events of pre-mRNA decay- and spliceosome-related genes.

miR778 mediates gene expression, histone modification, and DNA methylation during cyst nematode parasitism.

Despite the known critical regulatory functions of microRNAs, histone modifications, and DNA methylation in reprograming plant epigenomes in response to pathogen infection, the molecular mechanisms underlying the tight coordination of these components remain poorly understood. Here, we show how Arabidopsis (Arabidopsis thaliana) miR778 coordinately modulates the root transcriptome, histone methylation, and DNA methylation via post-transcriptional regulation of the H3K9 methyltransferases SU(var)3-9 homolog 5 (SUVH5) and SUVH6 upon infection by the beet cyst nematode Heterodera schachtii. miR778 post-transcriptionally silences SUVH5 and SUVH6 upon nematode infection. Manipulation of the expression of miR778 and its two target genes significantly altered plant susceptibility to H. schachtii. RNA-seq analysis revealed a key role of SUVH5 and SUVH6 in reprograming the transcriptome of Arabidopsis roots upon H. schachtii infection. In addition, chromatin immunoprecipitation (ChIP)-seq analysis established SUVH5 and SUVH6 as the main enzymes mediating H3K9me2 deposition in Arabidopsis roots in response to nematode infection. ChIP-seq analysis also showed that these methyltransferases possess distinct DNA binding preferences in that they are targeting transposable elements under noninfected conditions and protein-coding genes in infected plants. Further analyses indicated that H3K9me2 deposition directed by SUVH5 and SUVH6 contributes to gene expression changes both in roots and in nematode feeding sites and preferentially associates with CG DNA methylation. Together, our results uncovered multi-layered epigenetic regulatory mechanisms coordinated by miR778 during Arabidopsis-H. schachtii interactions.

Interactions of gene expression, alternative splicing, and DNA methylation in determining nodule identity.

Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules.

An Effector from the Cyst Nematode Heterodera schachtii Derepresses Host rRNA Genes by Altering Histone Acetylation.

Cyst nematodes are plant-pathogenic animals that secrete effector proteins into plant root cells to alter host gene expression and reprogram these cells to form specialized feeding sites, known as syncytia. The molecular mechanisms of these effectors are mostly unknown. We determined that the sugar beet cyst nematode (Heterodera schachtii) 32E03 effector protein strongly inhibits the activities of Arabidopsis thaliana histone deacetylases including the HDT1 enzyme, which has a known function in the regulation of rRNA gene expression through chromatin modifications. We determined that plants expressing the 32E03 coding sequence exhibited increased acetylation of histone H3 along the rDNA chromatin. At low 32E03 expression levels, these chromatin changes triggered the derepression of a subset of rRNA genes, which were conducive to H. schachtii parasitism. By contrast, high levels of 32E03 caused profound bidirectional transcription along the rDNA, which triggered rDNA-specific small RNA production leading to RNA-directed DNA methylation and silencing of rDNA, which inhibited nematode development. Our data show that the 32E03 effector alters plant rRNA gene expression by modulating rDNA chromatin in a dose-dependent manner. Thus, the 32E03 effector epigenetically regulates plant gene expression to promote cyst nematode parasitism.

Expression Patterns of DNA Methylation and Demethylation Genes during Plant Development and in Response to Phytohormones.

DNA methylation and demethylation precisely and effectively modulate gene expression during plant growth and development and in response to stress. However, expression profiles of genes involved in DNA methylation and demethylation during plant development and their responses to phytohormone treatments remain largely unknown. We characterized the spatiotemporal expression patterns of genes involved in de novo methylation, methyl maintenance, and active demethylation in roots, shoots, and reproductive organs using ß-glucuronidase (GUS) reporter lines. Promoters of DNA demethylases were generally more highly active at the mature root tissues, whereas the promoters of genes involved in DNA methylation were more highly active at fast-growing root tissues. The promoter activity also implies that methylation status in shoot apex, leaf primordia, floral organs, and developing embryos is under tight equilibrium through the activity of genes involved in DNA methylation and demethylation. The promoter activity of DNA methylation and demethylation-related genes in response to various phytohormone treatments revealed that phytohormones can alter DNA methylation status in specific and redundant ways. Overall, our results illustrate that DNA methylation and demethylation pathways act synergistically and antagonistically in various tissues and in response to phytohormone treatments and point to the existence of hormone-linked methylome regulation mechanisms that may contribute to tissue differentiation and development.

Re-targeting of a plant defense protease by a cyst nematode effector.

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector-coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two-hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain-like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity-based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re-localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two-hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector-mediated re-localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense-inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.

Identification of Differentially Methylated miRNA Genes During Compatible and Incompatible Interactions Between Soybean and Soybean Cyst Nematode.

DNA methylation is a widespread epigenetic mark that affects gene expression and transposon mobility during plant development and stress responses. However, the role of DNA methylation in regulating the expression of microRNA (miRNA) genes remains largely unexplored. Here, we analyzed DNA methylation changes of miRNA genes using a pair of soybean (Glycine max) near-isogenic lines (NILs) differing in their response to soybean cyst nematode (SCN; Heterodera glycines). Differences in global DNA methylation levels over miRNA genes in response to SCN infection were observed between the isogenic lines. miRNA genes with significant changes in DNA methylation levels in the promoter and primary transcript-coding regions were detected in both lines. In the susceptible isogenic line (NIL-S), 82 differentially methylated miRNAs were identified in response to SCN infection whereas, in the resistant isogenic line (NIL-R), only 16 differentially methylated miRNAs were identified. Interestingly, gma-miR5032, gma-miR5043, gma-miR1520b, and gma-2107-ch16 showed opposite methylation patterns in the isogenic lines. In addition, the miRNA paralogs gma-miR5770a and gma-miR5770b showed hypermethylation and hypomethylation in NIL-S and NIL-R, respectively. Gene expression quantification of gma-miR5032, gma-miR5043, gma-miR1520b, and gma-miR5770a/b and their confirmed targets indicated a role of DNA methylation in regulating miRNA expression and, thus, their targets upon SCN infection. Furthermore, overexpression of these four miRNAs in NIL-S using transgenic hairy root system enhanced plant resistance to SCN to various degrees with a key role observed for miR5032. Together, our results provide new insights into the role of epigenetic mechanisms in controlling miRNA regulatory function during SCN-soybean interactions.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A pathogenesis-related protein GmPR08-Bet VI promotes a molecular interaction between the GmSHMT08 and GmSNAP18 in resistance to Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is the most devastating pest affecting soybean production worldwide. SCN resistance requires both the GmSHMT08 and the GmSNAP18 in 'Peking'-type resistance. Here, we describe the molecular interaction between GmSHMT08 and GmSNAP18, which is potentiated by a pathogenesis-related protein GmPR08-Bet VI. Like GmSNAP18 and GmSHMT08, GmPR08-Bet VI expression was induced in response to SCN and its overexpression decreased SCN cysts by 65% in infected transgenic soybean roots. Overexpression of GmPR08-Bet VI did not have an effect on SCN resistance when the two cytokinin-binding sites in GmPR08-Bet VI were mutated, indicating a new role of GmPR08-Bet VI in SCN resistance. GmPR08-Bet VI was mapped to a QTL for resistance to SCN using different mapping populations. GmSHMT08, GmSNAP18 and GmPR08-Bet VI localize to the cytosol and plasma membrane. GmSNAP18 expression and localization hyper-accumulated at the plasma membrane and was specific to the root cells surrounding the nematode in SCN-resistant soybeans. Genes encoding key components of the salicylic acid signalling pathway were induced under SCN infection. GmSNAP18 and GmPR08-Bet VI were also induced under salicylic acid and cytokinin exogenous treatments, while GmSHMT08 was induced only when the resistant GmSNAP18 was present, pointing to the presence of a molecular crosstalk between SCN-resistant genes and defence genes. Expression analysis of GmSHMT08 and GmSNAP18 identified the need of a minimum expression requirement to trigger the SCN resistance reaction. These results provide insight into a new response mechanism towards plant nematode resistance involving haplotype compatibility, gene dosage and hormone signalling.

Identification of introduced and stably inherited DNA methylation variants in soybean associated with soybean cyst nematode parasitism.

DNA methylation is a widespread epigenetic mark that contributes to transcriptome reprogramming during plant-pathogen interactions. However, the distinct role of DNA methylation in establishing resistant and susceptible responses remains largely unexplored. Here, we developed and used a pair of near-isogenic lines (NILs) to characterize DNA methylome landscapes of soybean roots during the susceptible and resistant interactions with soybean cyst nematode (SCN; Heterodera glycines). We also compared the methylomes of the NILs and their parents to identify introduced and stably inherited methylation variants. The genomes of the NILs were substantially differentially methylated under uninfected conditions. This difference was associated with differential gene expression that may prime the NIL responses to SCN infection. In response to SCN infection, the susceptible line exhibited reduced global methylation levels in both protein-coding genes and transposable elements, whereas the resistant line showed the opposite response, increased global methylation levels. Heritable and novel nonparental differentially methylated regions overlapping with genes associated with soybean response to SCN infection were identified and validated using transgenic hairy root system. Our analyses indicate that DNA methylation patterns associated with the susceptible and resistant interactions are highly specific and that novel and stably inherited methylation variants are of biological significance.

A role for Arabidopsis growth-regulating factors 1 and 3 in growth-stress antagonism.

Growth-regulating factors (GRFs) belong to a small family of transcription factors that are highly conserved in plants. GRFs regulate many developmental processes and plant responses to biotic and abiotic stimuli. Despite the importance of GRFs, a detailed mechanistic understanding of their regulatory functions is still lacking. In this study, we used ChIP sequencing (ChIP-seq) to identify genome-wide binding sites of Arabidopsis GRF1 and GRF3, and correspondingly their direct downstream target genes. RNA-sequencing (RNA-seq) analysis revealed that GRF1 and GRF3 regulate the expression of a significant number of the identified direct targets. The target genes unveiled broad regulatory functions of GRF1 and GRF3 in plant growth and development, phytohormone biosynthesis and signaling, and the cell cycle. Our analyses also revealed that clock core genes and genes with stress- and defense-related functions are most predominant among the GRF1- and GRF3-bound targets, providing insights into a possible role for these transcription factors in mediating growth-defense antagonism and integrating environmental stimuli into developmental programs. Additionally, GRF1 and GRF3 target molecular nodes of growth-defense antagonism and modulate the levels of defense- and development-related hormones in opposite directions. Taken together, our results point to GRF1 and GRF3 as potential key determinants of plant fitness under stress conditions.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating plant-parasitic nematode. Most commercial soybean varieties with SCN resistance are derived from PI88788. Resistance derived from PI88788 is breaking down due to narrow genetic background and SCN population shift. PI88788 requires mainly the rhg1-b locus, while 'Peking' requires rhg1-a and Rhg4 for SCN resistance. In the present study, whole genome re-sequencing of 106 soybean lines was used to define the Rhg haplotypes and investigate their responses to the SCN HG-Types. The analysis showed a comprehensive profile of SNPs and copy number variations (CNV) at these loci. CNV of rhg1 (GmSNAP18) only contributed towards resistance in lines derived from PI88788 and 'Cloud'. At least 5.6 copies of the PI88788-type rhg1 were required to confer SCN resistance, regardless of the Rhg4 (GmSHMT08) haplotype. However, when the GmSNAP18 copies dropped below 5.6, a 'Peking'-type GmSHMT08 haplotype was required to ensure SCN resistance. This points to a novel mechanism of epistasis between GmSNAP18 and GmSHMT08 involving minimum requirements for copy number. The presence of more Rhg4 copies confers resistance to multiple SCN races. Moreover, transcript abundance of the GmSHMT08 in root tissue correlates with more copies of the Rhg4 locus, reinforcing SCN resistance. Finally, haplotype analysis of the GmSHMT08 and GmSNAP18 promoters inferred additional levels of the resistance mechanism. This is the first report revealing the genetic basis of broad-based resistance to SCN and providing new insight into epistasis, haplotype-compatibility, CNV, promoter variation and its impact on broad-based disease resistance in plants.

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly.

BACKGROUND: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. RESULTS: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. CONCLUSION: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly.

Canonical and noncanonical ethylene signaling pathways that regulate Arabidopsis susceptibility to the cyst nematode Heterodera schachtii.

Plant-parasitic cyst nematodes successfully exploit various phytohormone signaling pathways to establish a new hormonal equilibrium that facilitates nematode parasitism. Although it is largely accepted that ethylene regulates plant responses to nematode infection, a mechanistic understanding of how ethylene shapes plant-nematode interactions remains largely unknown. In this study, we examined the involvement of various components regulating ethylene perception and signaling in establishing Arabidopsis susceptibility to the cyst nematode Heterodera schachtii using a large set of well-characterized single and higher order mutants. Our analyses revealed the existence of two pathways that separately engage ethylene with salicylic acid (SA) and cytokinin signaling during plant response to nematode infection. One pathway involves the canonical ethylene signaling pathway in which activation of ethylene signaling results in suppression of SA-based immunity. The second pathway involves the ethylene receptor ETR1, which signals independently of SA acid to affect immunity, instead altering cytokinin-mediated regulation of downstream components. Our results reveal important mechanisms through which cyst nematodes exploit components of ethylene perception and signaling to affect the balance of hormonal signaling through ethylene interaction with SA and cytokinin networks. This hormonal interaction overcomes plant defense and provokes a susceptible response.

Ethylene Receptors Signal via a Noncanonical Pathway to Regulate Abscisic Acid Responses.

Ethylene is a gaseous plant hormone perceived by a family of receptors in Arabidopsis (Arabidopsis thaliana) including ETHYLENE RESPONSE1 (ETR1) and ETR2. Previously we showed that etr1-6 loss-of-function plants germinate better and etr2-3 loss-of-function plants germinate worse than wild-type under NaCl stress and in response to abscisic acid (ABA). In this study, we expanded these results by showing that ETR1 and ETR2 have contrasting roles in the control of germination under a variety of inhibitory conditions for seed germination such as treatment with KCl, CuSO4, ZnSO4, and ethanol. Pharmacological and molecular biology results support a model where ETR1 and ETR2 are indirectly affecting the expression of genes encoding ABA signaling proteins to affect ABA sensitivity. The receiver domain of ETR1 is involved in this function in germination under these conditions and controlling the expression of genes encoding ABA signaling proteins. Epistasis analysis demonstrated that these contrasting roles of ETR1 and ETR2 do not require the canonical ethylene signaling pathway. To explore the importance of receptor-protein interactions, we conducted yeast two-hybrid screens using the cytosolic domains of ETR1 and ETR2 as bait. Unique interacting partners with either ETR1 or ETR2 were identified. We focused on three of these proteins and confirmed the interactions with receptors. Loss of these proteins led to faster germination in response to ABA, showing that they are involved in ABA responses. Thus, ETR1 and ETR2 have both ethylene-dependent and -independent roles in plant cells that affect responses to ABA.

Homeostasis in the soybean miRNA396-GRF network is essential for productive soybean cyst nematode infections.

Heterodera glycines, the soybean cyst nematode, penetrates soybean roots and migrates to the vascular cylinder where it forms a feeding site called the syncytium. MiRNA396 (miR396) targets growth-regulating factor (GRF) genes, and the miR396-GRF1/3 module is a master regulator of syncytium development in model cyst nematode H. schachtii infection of Arabidopsis. Here, we investigated whether this regulatory system operates similarly in soybean roots and is likewise important for H. glycines infection. We found that a network involving nine MIR396 and 23 GRF genes is important for normal development of soybean roots and that GRF function is specified in the root apical meristem by miR396. All MIR396 genes are down-regulated in the syncytium during its formation phase while, specifically, 11 different GRF genes are up-regulated. The switch to the syncytium maintenance phase coincides with up-regulation of MIR396 and down-regulation of the 11 GRF genes specifically via post-transcriptional regulation by miR396. Furthermore, interference with the miR396-GRF6/8-13/15-17/19 regulatory network, through either overexpression or knockdown experiments, does not affect the number of H. glycines juveniles that enter the vascular cylinder to initiate syncytia, but specifically inhibits efficient H. glycines development to adult females. Therefore, homeostasis in the miR396-GRF6/8-13/15-17/19 regulatory network is essential for productive H. glycines infections.

A novel picornavirus-like genome from transcriptome sequencing of sugar beet cyst nematode represents a new putative genus.

Analysis of transcriptome sequence data from eggs and second-stage juveniles (J2s) of sugar beet cyst nematode (SBCN, Heterodera schachtii) identified the full-length genome of a positive-sense single-stranded RNA virus, provisionally named sugar beet cyst nematode virus 1 (SBCNV1). The SBCNV1 sequence was detected in both eggs and J2s, indicating its possible vertical transmission. The 9503-nucleotide genome sequence contains a single long open reading frame, which was predicted to encode a polyprotein with conserved domains for picornaviral structural proteins proximal to its amino terminus and RNA helicase, cysteine proteinase and RNA-dependent RNA polymerase (RdRp) conserved domains proximal to its carboxyl terminus, hallmarks of viruses belonging to the order Picornavirales. Phylogenetic analysis of the predicted SBCNV1 RdRp amino acid sequence indicated that the SBCNV1 sequence is most closely related to members of the family Secoviridae, which includes genera of nematode-transmitted plant-infecting viruses. SBCNV1 represents the first fully sequenced viral genome from SBCN.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.

Cyst Nematode Parasitism Induces Dynamic Changes in the Root Epigenome.

A growing body of evidence indicates that epigenetic modifications can provide efficient, dynamic, and reversible cellular responses to a wide range of environmental stimuli. However, the significance of epigenetic modifications in plant-pathogen interactions remains largely unexplored. In this study, we provide a comprehensive analysis of epigenome changes during the compatible interaction between the beet cyst nematode Heterodera schachtii and Arabidopsis (Arabidopsis thaliana). Whole-genome bisulfite sequencing was conducted to assess the dynamic changes in the methylome of Arabidopsis roots in response to H. schachtii infection. H. schachtii induced widespread hypomethylation of protein-coding genes and transposable elements (TEs), preferentially those adjacent to protein-coding genes. The abundance of 24-nt siRNAs was associated with hypermethylation of TEs and gene promoters, with influence observed for methylation context and infection time points. mRNA sequencing revealed a significant enrichment for the differentially methylated genes among the differentially expressed genes, specifically those with functions corresponding to primary metabolic processes and responses to stimuli. The differentially methylated genes overlapped with more than one-fourth of the syncytium differentially expressed genes and are of functional significance. Together, our results provide intriguing insights into the potential regulatory role of differential DNA methylation in shaping the biological interplay between cyst nematodes and host plants.