Transcriptomic changes in soybean underlying growth promotion and defense against cyst nematode after Bacillus simplex Sneb545 treatment.

Bacillus simplex Sneb45 is a plant-growth-promoting rhizobacterium that promotes soybean growth and systemic resistance to cyst nematode. To investigate transcriptional changes in soybean roots in response to B. simplex Sneb45 treatment, transcriptome analysis and quantitative real-time PCR were conducted to detect and validate the differentially expressed genes (DEGs). In total, 19,109 DEGs were obtained. After B. simplex Sneb545 treatment, 970 and 1265 genes were up- and down-regulated at 5 days post-inoculation (dpi), respectively, and 142 and 47 genes were up- and down-regulated at 10 dpi, respectively, compared with untreated soybean roots. Functional annotation of DEGs indicated that B. simplex Sneb545 regulated soybean growth and defense against cyst nematode possibly through genes related to auxin, gibberellin, and NB-LRR protein. In addition, GO and KEGG enrichment analyses indicated that the DEGs were enriched in metabolism, signal transduction, and plant-pathogen interaction pathways. Moreover, the auxin and gibberellin contents were lower in B. simplex Sneb545-treated soybean roots than in untreated roots at 5 dpi. B. simplex Sneb545 possibly altered the expression of wound-induced protein and NAC transcription factor to regulate soybean growth and defense against cyst nematode. Our study provided deep insights into the alterations in soybean transcriptome after exposure to B. simplex Sneb45 and a theoretical basis for further exploring molecular functions underlying the biological control activity of B. simplex Sneb545.

Transcriptome Analysis of GmPUB20A Overexpressing and RNA-Interferencing Transgenic Hairy Roots Reveals Underlying Negative Role in Soybean Resistance to Cyst Nematode.

Ubiquitination genes are key components of plant responses to biotic stress. GmPUB20A, a ubiquitination gene, plays a negative role in soybean resistance to soybean cyst nematode (SCN). In this study, we employed high-throughput sequencing to investigate transcriptional changes in GmPUB20A overexpressing and RNA-interfering transgenic hairy roots. Totally, 7661 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that DEGs were significantly enriched in disease resistance and signal transduction pathways. In addition, silencing Glyma.15G021600 and Glyma.09G284700 by siRNA, the total number of nematodes was decreased by 33.48% and 27.47% than control plants, respectively. Further, GUS activity and reactive oxygen species (ROS) assays revealed that GmPUB20A, Glyma.15G021600, and Glyma.09G284700 respond to SCN parasitism and interfere with the accumulation of ROS in plant roots, respectively. Collectively, our study provides insights into the molecular mechanism of GmPUB20A in soybean resistance to SCN.

Gma-miR408 Enhances Soybean Cyst Nematode Susceptibility by Suppressing Reactive Oxygen Species Accumulation.

Soybean cyst nematode (SCN, Heterodera glycine) is a serious damaging disease in soybean worldwide, thus resulting in severe yield losses. MicroRNA408 (miR408) is an ancient and highly conserved miRNA involved in regulating plant growth, development, biotic and abiotic stress response. Here, we analyzed the evolution of miR408 in plants and verified four miR408 members in Glycine max. In the current research, highly upregulated gma-miR408 expressing was detected during nematode migration and syncytium formation response to soybean cyst nematode infection. Overexpressing and silencing miR408 vectors were transformed to soybean to confirm its potential role in plant and nematode interaction. Significant variations were observed in the MAPK signaling pathway with low OXI1, PR1, and wounding of the overexpressing lines. Overexpressing miR408 could negatively regulate soybean resistance to SCN by suppressing reactive oxygen species accumulation. Conversely, silencing miR408 positively regulates soybean resistance to SCN. Overall, gma-miR408 enhances soybean cyst nematode susceptibility by suppressing reactive oxygen species accumulation.

Functional Characterization of Ubiquitination Genes in the Interaction of Soybean-Heterodera glycines.

Ubiquitination is a kind of post-translational modification of proteins that plays an important role in plant response to biotic and abiotic stress. The response of soybean GmPUB genes to soybean cyst nematode (SCN, Heterodera glycines) infection is largely unknown. In this study, quantitative real-time PCR (qRT-PCR) was performed to detect the relative expression of 49 GmPUB genes in susceptible cultivar William 82 and resistant cultivar Huipizhi after SCN inoculation. The results show that GmPUB genes responded to cyst nematode infection at 1 day post-inoculation (dpi), 5 dpi, 10 dpi and 15 dpi. The expression levels of GmPUB16A, GmPUB20A, GmCHIPA, GmPUB33A, GmPUB23A and GmPUB24A were dramatically changed during SCN infection. Furthermore, functional analysis of these GmPUB genes by overexpression and RNAi showed that GmPUB20A, GmPUB33A and GmPUB24A negatively regulated soybean resistance under SCN stress. The results from our present study provide insights into the complicated molecular mechanism of the interaction between soybean and SCN.

Genome-wide identification of small interfering RNAs from sRNA libraries constructed from soybean cyst nematode resistant and susceptible cultivars.

Plant small-RNAs regulate various biological processes by manipulating the expression of target genes at the transcriptional and post-transcriptional levels. However, little is known about the response and the functional roles of sRNAs, particularly small-interfering RNAs (siRNAs), in the soybean-soybean cyst nematode interaction. In this study, siRNA data from 24 sRNA libraries constructed from SCN-infected and non-SCN-infected resistant and susceptible soybean roots were analysed in silico. A total of 26 novel siRNAs including 17 phasiRNAs and 9 nat-siRNAs, as well as two phasiRNAs that were differentially expressed (DE) in three comparisons, were identified. Then, using qRT-PCR, the expression of majority of siRNAs was found to be downregulated after SCN infection, and the expression patterns of DE siRNAs were confirmed. Further functional annotation analyses revealed that the target genes of these siRNA were highly related to disease resistance, which included the genes coding for the NB-ARC domain, leucine-rich repeats, and Hs1pro-1 homologous proteins. Overall, the present research identified novel siRNAs and annotated their target genes, thereby laying the foundation for deciphering the roles of siRNAs in the soybean-SCN interaction.

Soybean miR159-GmMYB33 Regulatory Network Involved in Gibberellin-Modulated Resistance to Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is an obligate sedentary biotroph that poses major threats to soybean production globally. Recently, multiple miRNAome studies revealed that miRNAs participate in complicated soybean-SCN interactions by regulating their target genes. However, the functional roles of miRNA and target genes regulatory network are still poorly understood. In present study, we firstly investigated the expression patterns of miR159 and targeted GmMYB33 genes. The results showed miR159-3p downregulation during SCN infection; conversely, GmMYB33 genes upregulated. Furthermore, miR159 overexpressing and silencing soybean hairy roots exhibited strong resistance and susceptibility to H. glycines, respectively. In particular, miR159-GAMYB genes are reported to be involve in GA signaling and metabolism. Therefore, we then investigated the effects of GA application on the expression of miR159-GAMYB module and the development of H. glycines. We found that GA directly controls the miR159-GAMYB module, and exogenous GA application enhanced endogenous biologically active GA1 and GA3, the abundance of miR159, lowered the expression of GmMYB33 genes and delayed the development of H. glycines. Moreover, SCN infection also results in endogenous GA content decreased in soybean roots. In summary, the soybean miR159-GmMYB33 module was directly involved in the GA-modulated soybean resistance to H. glycines.

Evaluation of Scopoletin from Penicillium janthinellum Snef1650 for the Control of Heterodera glycines in Soybean.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is responsible for causing a major soybean disease globally. The fungal strain Penicillium janthinellum Snef1650 was evaluated against H. glycines. However, the effective determinants of the P. janthinellum strain are unknown. By performing pot experiments, a functioning compound was isolated from P. janthinellum Snef1650 through organic solvent extraction, semi-preparative HPLC, Sephadex LH-20 column chromatography, and silica gel column chromatography, and the isolated compound was identified to be scopoletin through 1H NMR, 13C NMR, and HPLC-MS. The pot experiments indicated that the treatment of soybean seeds with scopoletin drastically reduced the SCN population. The field experiments performed in 2017 and 2018 revealed that scopoletin decreased over 43.7% juveniles in the roots and over 61.55% cysts in the soil. Scopoletin treatment also promoted soybean growth and improved its yield, with an increase in plot yield by >5.33%. Scopoletin obtained from P. janthinellum Snef1650 could be used as an anti-H. glycines biocontrol agent.

Identification of MicroRNAs That Respond to Soybean Cyst Nematode Infection in Early Stages in Resistant and Susceptible Soybean Cultivars.

Soybean cyst nematode (SCN) causes heavy losses to soybean yield. In order to investigate the roles of soybean miRNAs during the early stages of infection (1 and 5 dpi), 24 small RNA libraries were constructed from SCN resistant cultivar Huipizhi (HPZ) and the susceptible Williams 82 (W82) cultivar for high-throughput sequencing. By sequencing the small RNA libraries, a total of 634 known miRNAs were identified, and 252 novel miRNAs were predicted. Altogether, 14 known miRNAs belonging to 13 families, and 26 novel miRNAs were differentially expressed and may respond to SCN infection in HPZ and W82. Similar expression results were also confirmed by qRT-PCR. Further analysis of the biological processes that these potential target genes of differentially expressed miRNAs regulate found that they may be strongly related to plant-pathogen interactions. Overall, soybean miRNAs experience profound changes in early stages of SCN infection in both HPZ and W82. The findings of this study can provide insight into miRNAome changes in both HPZ and W82 at the early stages of infection, and may provide a stepping stone for future SCN management.