Genetic characterization of qSCN10 from an exotic soybean accession PI 567516C reveals a novel source conferring broad-spectrum resistance to soybean cyst nematode.

KEY MESSAGE: The qSCN10 locus with broad-spectrum SCN resistance was fine-mapped to a 379-kb region on chromosome 10 in soybean accession PI 567516C. Candidate genes and potential application benefits of this locus were discussed. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most devastating pests of soybean, causing significant yield losses worldwide every year. Genetic resistance has been the major strategy to control this pest. However, the overuse of the same genetic resistance derived primarily from PI 88788 has led to the genetic shifts in nematode populations and resulted in the reduced effectiveness in soybean resistance to SCN. Therefore, novel genetic resistance resources, especially those with broad-spectrum resistance, are needed to develop new resistant cultivars to cope with the genetic shifts in nematode populations. In this study, a quantitative trait locus (QTL) qSCN10 previously identified from a soybean landrace PI 567516C was confirmed to confer resistance to multiple SCN HG Types. This QTL was further fine-mapped to a 379-kb region. There are 51 genes in this region. Four of them are defense-related and were regulated by SCN infection, suggesting their potential role in mediating resistance to SCN. The phylogenetic and haplotype analyses of qSCN10 as well as other information indicate that this locus is different from other reported resistance QTL or genes. There was no yield drag or other unfavorable traits associated with this QTL when near-isogenic lines with and without qSCN10 were tested in a SCN-free field. Therefore, our study not only provides further insight into the genetic basis of soybean resistance to SCN, but also identifies a novel genetic resistance resource for breeding soybean for durable, broad-spectrum resistance to this pest.

Fine-mapping and characterization of qSCN18, a novel QTL controlling soybean cyst nematode resistance in PI 567516C.

KEY MESSAGE: The qSCN18 QTL from PI 56756C was confirmed and fine-mapped to improve soybean resistance to the SCN population HG Type 2.5.7 using near-isogenic lines carrying recombination crossovers within the QTL region. The QTL underlying resistance was fine-mapped to a 166-Kbp region on chromosome 18, and the candidate genes were selected based on genomic analyses. Soybean cyst nematode (SCN, Heterodera glycines, Ichinohe) is the most devastating pathogen of soybean. Understanding the genetic basis of SCN resistance is crucial for managing this parasite in the field. Two major loci, rhg1 and Rhg4, were previously characterized as valuable resources for SCN resistance. However, their continuous use has caused shifts in the virulence of SCN populations, which can overcome the resistance conferred by these two major loci. Reduced effectiveness became a major concern in the soybean industry due to continuous use of rhg1 for decades. Thus, it is imperative to identify sources of SCN resistance for durable SCN management. A novel QTL qSCN18 was identified in PI567516C. To fine-map qSCN18 and identify resistance genes, a large backcross population was developed. Nineteen near-isogenic lines (NILs) carrying recombination crossovers within the QTL region were identified. The first phase of fine-mapping narrowed the QTL region to 549-Kbp, whereas the second phase confined the region to 166-Kbp containing 23 genes. Two flanking markers, MK-1 and MK-6, were developed and validated to detect the presence of the qSCN18 resistance allele. Haplotype analysis clustered the fine-mapped qSCN18 region from PI 567516C with the cqSCN-007 locus previously mapped in the wild soybean accession PI 468916. The NILs were developed to further characterize the causal gene(s) harbored in this QTL. This study also confirmed the previously identified qSCN18. The results will facilitate marker-assisted selection (MAS) introducing the qSCN18 locus from PI 567516C into high-yielding soybean cultivars with durable resistance to SCN.

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.

Genomic-assisted phylogenetic analysis and marker development for next generation soybean cyst nematode resistance breeding.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is a serious soybean pest. The use of resistant cultivars is an effective approach for preventing yield loss. In this study, 19,652 publicly available soybean accessions that were previously genotyped with the SoySNP50K iSelect BeadChip were used to evaluate the phylogenetic diversity of SCN resistance genes Rhg1 and Rhg4 in an attempt to identify novel sources of resistance. The sequence information of soybean lines was utilized to develop KASPar (KBioscience Competitive Allele-Specific PCR) assays from single nucleotide polymorphisms (SNPs) of Rhg1, Rhg4, and other novel quantitative trait loci (QTL). These markers were used to genotype a diverse set of 95 soybean germplasm lines and three recombinant inbred line (RIL) populations. SNP markers from the Rhg1 gene were able to differentiate copy number variation (CNV), such as resistant-high copy (PI 88788-type), low copy (Peking-type), and susceptible-single copy (Williams 82) numbers. Similarly, markers for the Rhg4 gene were able to detect Peking-type (resistance) genotypes. The phylogenetic information of SCN resistance loci from a large set of soybean accessions and the gene/QTL specific markers that were developed in this study will accelerate SCN resistance breeding programs.

Whole-genome gene expression profiling revealed genes and pathways potentially involved in regulating interactions of soybean with cyst nematode (Heterodera glycines Ichinohe).

BACKGROUND: Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most devastating pathogen of soybean. Many gene expression profiling studies have been conducted to investigate the responses of soybean to the infection by this pathogen using primarily the first-generation soybean genome array that covered approximately 37,500 soybean transcripts. However, no study has been reported yet using the second-generation Affymetrix soybean whole-genome transcript array (Soybean WT array) that represents approximately 66,000 predicted soybean transcripts. RESULTS: In the present work, the gene expression profiles of two soybean plant introductions (PIs) PI 437654 and PI 567516C (both resistant to multiple SCN HG Types) and cultivar Magellan (susceptible to SCN) were compared in the presence or absence of the SCN inoculum at 3 and 8 days post-inoculation using the Soybean WT array. Data analysis revealed that the two resistant soybean lines showed distinctive gene expression profiles from each other and from Magellan not only in response to the SCN inoculation, but also in the absence of SCN. Overall, 1,413 genes and many pathways were revealed to be differentially regulated. Among them, 297 genes were constitutively regulated in the two resistant lines (compared with Magellan) and 1,146 genes were responsive to the SCN inoculation in the three lines, with 30 genes regulated both constitutively and by SCN. In addition to the findings similar to those in the published work, many genes involved in ethylene, protein degradation, and phenylpropanoid pathways were also revealed differentially regulated in the present study. GC-rich elements (e.g., GCATGC) were found over-represented in the promoter regions of certain groups of genes. These have not been observed before, and could be new defense-responsive regulatory elements. CONCLUSIONS: Different soybean lines showed different gene expression profiles in the presence and absence of the SCN inoculum. Both inducible and constitutive gene expression may contribute to resistance to multiple SCN HG Types in the resistant soybean PI lines. Ethylene, protein degradation, and phenylpropanoid pathways, as well as many other pathways reported previously, may play important roles in mediating the soybean-SCN interactions. The revealed genes, pathways, and promoter elements can be further explored to regulate or engineer soybean for resistance to SCN.

Pinpointing genes underlying the quantitative trait loci for root-knot nematode resistance in palaeopolyploid soybean by whole genome resequencing.

The objective of this study was to use next-generation sequencing technologies to dissect quantitative trait loci (QTL) for southern root-knot nematode (RKN) resistance into individual genes in soybean. Two hundred forty-six recombinant inbred lines (RIL) derived from a cross between Magellan (susceptible) and PI 438489B (resistant) were evaluated for RKN resistance in a greenhouse and sequenced at an average of 0.19× depth. A sequence analysis pipeline was developed to identify and validate single-nucleotide polymorphisms (SNPs), infer the parental source of each SNP allele, and genotype the RIL population. Based on 109,273 phased SNPs, recombination events in RILs were identified, and a total of 3,509 bins and 3,489 recombination intervals were defined. About 50.8% of bins contain 1 to 10 genes. A linkage map was subsequently constructed by using bins as molecular markers. Three QTL for RKN resistance were identified. Of these, one major QTL was mapped to bin 10 of chromosome 10, which is 29.7 kb in size and harbors three true genes and two pseudogenes. Based on sequence variations and gene-expression analysis, the candidate genes underlying the major QTL for RKN resistance were pinpointed. They are Glyma10g02150 and Glyma10g02160, encoding a pectin methylesterase inhibitor and a pectin methylesterase inhibitor -pectin methylesterase, respectively. This QTL mapping approach not only combines SNP discovery, SNP validation, and genotyping, but also solves the issues caused by genome duplication and repetitive sequences. Hence, it can be widely used in crops with a reference genome to enhance QTL mapping accuracy.

LYK4, a lysin motif receptor-like kinase, is important for chitin signaling and plant innate immunity in Arabidopsis.

Chitin is commonly found in fungal cell walls and is one of the well-studied microbe/pathogen-associated molecular patterns. Previous studies showed that lysin motif (LysM)-containing proteins are essential for plant recognition of chitin, leading to the activation of plant innate immunity. In Arabidopsis (Arabidopsis thaliana), the LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1) was shown to be essential for chitin recognition, whereas in rice (Oryza sativa), the LysM-containing protein, CEBiP (for chitin elicitor-binding protein), was shown to be involved in chitin recognition. Unlike LYK1/CERK1, CEBiP lacks an intracellular kinase domain. Arabidopsis possesses three CEBiP-like genes. Our data show that mutations in these genes, either singly or in combination, did not compromise the response to chitin treatment. Arabidopsis also contains five LYK genes. Analysis of mutations in LYK2, -3, -4, or -5 showed that LYK4 is also involved in chitin signaling. The lyk4 mutants showed reduced induction of chitin-responsive genes and diminished chitin-induced cytosolic calcium elevation as well as enhanced susceptibility to both the bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the fungal pathogen Alternaria brassicicola, although these phenotypes were not as dramatic as that seen in the lyk1/cerk1 mutants. Similar to LYK1/CERK1, the LYK4 protein was also localized to the plasma membrane. Therefore, LYK4 may play a role in the chitin recognition receptor complex to assist chitin signal transduction and plant innate immunity.

Ethylene-responsive element-binding factor 5, ERF5, is involved in chitin-induced innate immunity response.

Our recent work demonstrated that chitin treatment modulated the expression of 118 transcription factor (TF) genes in Arabidopsis. To investigate the potential roles of these TF in chitin signaling and plant defense, we initiated an interaction study among these TF proteins, as well as two chitin-activated mitogen-activated protein kinases (MPK3 and MPK6), using a yeast two-hybrid system. This study revealed interactions among the following proteins: three ethylene-responsive element-binding factors (ERF), five WRKY transcription factors, one scarecrow-like (SCL), and the two MPK, in addition to many other interactions, reflecting a complex TF interaction network. Most of these interactions were subsequently validated by other methods, such as pull-down and in planta bimolecular fluorescence complementation assays. The key node ERF5 was shown to interact with multiple proteins in the network, such as ERF6, ERF8, and SCL13, as well as MPK3 and MPK6. Interestingly, ERF5 appeared to negatively regulate chitin signaling and plant defense against the fungal pathogen Alternaria brassicicola and positively regulate salicylic acid signaling and plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Therefore, ERF5 may play an important role in plant innate immunity, likely through coordinating chitin and other defense pathways in plants in response to different pathogens.

Discovery of ADP-ribosylation and other plant defense pathway elements through expression profiling of four different Arabidopsis-Pseudomonas R-avr interactions.

A dissection of plant defense pathways was initiated through gene expression profiling of the responses of a single Arabidopsis thaliana genotype to isogenic Pseudomonas syringae strains expressing one of four different cloned avirulence (avr) genes. Differences in the expression profiles elicited by different resistance (R)-avr interactions were observed. A role for poly(ADP-ribosyl)ation in plant defense responses was suggested initially by the upregulated expression of genes encoding NUDT7 and poly(ADP-ribose) glycohydrolase in multiple R-avr interactions. Gene knockout plant lines were tested for 20 candidate genes identified by the expression profiling, and Arabidopsis NUDT7 mutants allowed less growth of virulent P. syringae (as previously reported) but also exhibited a reduced hypersensitive-response phenotype. Inhibitors of poly(ADP-ribose) polymerase (PARP) disrupted FLS2-mediated basal defense responses such as callose deposition. EIN2 (ethylene response) and IXR1 and IXR2 (cellulose synthase) mutants impacted the FLS2-mediated responses that occur during PARP inhibition, whereas no impacts were observed for NPR1, PAD4, or NDR1 mutants. In the expression profiling work, false-positive selection and grouping of genes was reduced by requiring simultaneous satisfaction of statistical significance criteria for each of three separate analysis methods, and by clustering genes based on statistical confidence values for each gene rather than on average fold-change of transcript abundance.

Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin.

SUMMARY Chitin, a polysaccharide composed of beta-1-->4-linked N-acetyl-d-glucosamine, has been shown or implicated as a signal in plant defence and development. However, the key components of chitin perception and downstream signalling in non-leguminous plants are largely unknown. In recent years, mitogen-activated protein kinases (MAPKs) and their cascades were shown to transduce various extracellular stimuli into internal cellular responses. To investigate the possible involvement of MAPKs in chitin signalling in plants, the model plant Arabidopsis thaliana was treated with crab-shell chitin and also with the purified chitin oligomers (degree of polymerization, d.p. = 2-8). Both mRNA levels and kinase activity of two MAPK genes, AtMPK6 and AtMPK3, were monitored after treatment. The mRNA of AtMPK3 was strongly up-regulated by both chitin and its larger oligomers (d.p. = 6-8), but the mRNA of AtMPK6 did not appear to be regulated by these treatments. However, the kinase activity of both MAPKs was induced by chitin and the larger oligomers (d.p. = 6-8), with AtMPK6 much more strongly induced. In addition, WRKY22, WRKY29, WRKY33 and WRKY53, which encode four WRKY transcription factors that recognize TTGAC(C/T) W-box elements in promoters of numerous plant defence-related genes, were up-regulated by these treatments. WRKY33 and WRKY53 expression was induced by the transgenic expression of the tobacco MAPKK NtMEK2 active mutant NtMEK2(DD), suggesting a potential role for these WRKY transcription factors in relaying the signal generated from the MAPK cascade to downstream genes. These data suggest that AtMPK6/AtMPK3 and WRKY transcription factors (such as WRKY33 and WRKY53) may be important components of a pathway involved in chitin signalling in Arabidopsis plants.