Time Course RNA-seq Reveals Soybean Responses against Root-Lesion Nematode and Resistance Players.

Pratylenchus brachyurus causes serious damage to soybean production and other crops worldwide. Plant molecular responses to RLN infection remain largely unknown and no resistance genes have been identified in soybean. In this study, we analyzed molecular responses to RLN infection in moderately resistant BRSGO (Chapadões-BRS) and susceptible TMG115 RR (TMG) Glycine max genotypes. Differential expression analysis revealed two stages of response to RLN infection and a set of differentially expressed genes (DEGs) in the first stage suggested a pattern-triggered immunity (PTI) in both genotypes. The divergent time-point of DEGs between genotypes was observed four days post-infection, which included the activation of mitogen-activated protein kinase (MAPK) and plant-pathogen interaction genes in the BRS, suggesting the occurrence of an effector-triggered immunity response (ETI) in BRS. The co-expression analyses combined with single nucleotide polymorphism (SNP) uncovered a key element, a transcription factor phytochrome-interacting factor (PIF7) that is a potential regulator of moderate resistance to RLN infection. Two genes for resistance-related leucine-rich repeat (LRR) proteins were found as BRS-specific expressed genes. In addition, alternative splicing analysis revealed an intron retention in a myo-inositol oxygenase (MIOX) transcript, a gene related to susceptibility, may cause a loss of function in BRS.

Genome-wide association study for resistance to the Meloidogyne javanica causing root-knot nematode in soybean.

KEY MESSAGE: A locus on chromosome 13, containing multiple TIR-NB-LRR genes and SNPs associated with M. javanica resistance, was identified using a combination of GWAS, resequencing, genetic mapping and expression profiling. Meloidogyne javanica, a root-knot nematode, is an important problem in soybean-growing areas, leading to severe yield losses. Some accessions have been identified carrying resistance loci to this nematode. In this study, a set of 317 soybean accessions was characterized for resistance to M. javanica. A genome-wide association study was performed using SNPs from genotyping-by-sequencing, and a region of 29.2 kb on chromosome 13 was identified. An analysis of haplotypes showed that SNPs were able to discriminate between susceptible and resistant accessions, with 25 accessions sharing the haplotype associated with resistance. Furthermore, five accessions that exhibited resistance without carrying this haplotype may carry different loci conferring resistance to M. javanica. We also conducted the screening of the SNPs in the USDA soybean germplasm, revealing that several soybean accessions previously reported as resistant to other nematodes also shared the resistance haplotype on chromosome 13. Two SNP-based TaqMan® assays were developed and validated in two panels of soybean cultivars and in biparental populations. In silico analysis of the region associated with resistance identified the occurrence of genes with structural similarity with classical major resistance genes (NBS-LRR genes). Specifically, several nonsynonymous SNPs were observed in Glyma.13g194800 and Glyma.13g194900. The expression profile of these candidate genes demonstrated that the two gene models were up-regulated in the resistance source PI 505,099 after nematode infection. Overall, the SNPs associated with resistance and the genes identified constitute an important tool for introgression of resistance to the root-knot nematode by marker-assisted selection in soybean breeding programs.

The soybean gene GmHsp22.4 is involved in the resistance response to Meloidogyne javanica in Arabidopsis thaliana.

BACKGROUND: Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. A previous study has showed strong induction of the gene GmHsp22.4 in response to the nematode Meloidogyne javanica in a resistant soybean genotype, while repression in a susceptible one. This study aimed to investigate the functional involvement of this small chaperone in response to M. javanica in Arabidopsis thaliana. First, it was evaluated the activation of the promoter region after the nematode inoculation, and the occurrence of polymorphisms between resistant and susceptible re-sequenced soybean accessions. Then functional analysis using A. thaliana lines overexpressing the soybean GmHsp22.4 gene, and knocked-out mutants were challenged with M. javanica infestation. RESULTS: High expression levels of the GFP gene marker in transformed A. thaliana plants revealed that the promoter region of GmHsp22.4 was strongly activated after nematode inoculation. Moreover, the multiplication of the nematode was significantly reduced in plants overexpressing GmHsp22.4 gene in A. thaliana compared to the wild type. Additionally, the multiplication of M. javanica in the A. thaliana mutants was significantly increased mainly in the event athsp22.0-2. This increase was not that evident in the event athsp22.0-1, the one that preserved a portion of the promoter region, including the HSEs in the region around - 83 bp. However, structural analysis at sequence level among soybean resistant and susceptible genotypes did not detect any polymorphisms in the whole gene model. CONCLUSIONS: The soybean chaperone GmHsp22.4 is involved in the defense response to root-knot nematode M. javanica in A. thaliana. Specifically, the promoter region covering until - 191 from the transcriptional start site (TSS) is necessary to promoter activation after nematode infection in Arabidopsis. No polymorphisms that could explain these differences in the defense response were detected in the GmHsp22.4 gene between resistant and susceptible soybean genotypes. Therefore, further investigation is needed to elucidate the triggering factor of the plant's defense mechanism, both at the sequence level of the soybean genotypes presenting contrasting reaction to root-knot nematode and by detecting cis-elements that are essential for the activation of the GmHsp22.4 gene promoter.

Crotalaria spectabilis as a source of pyrrolizidine alkaloids and phenolic compounds: HPLC-MS/MS dereplication and monocrotaline quantification of seed and leaf extracts.

INTRODUCTION: Crotalaria spectabilis is an important species used as a pre-plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under-explored. OBJECTIVE: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. MATERIALS AND METHODS: Seeds and leaves extracts were analysed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification of the compounds. RESULTS: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra-high-performace liquid chromatography-quadrupole time-of-flight (UHPLC-QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC-MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. CONCLUSION: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts.