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.

Interaction between Rag genes results in a unique synergistic transcriptional response that enhances soybean resistance to soybean aphids

Background Pyramiding different resistance genes into one plant genotype confers enhanced resistance at the phenotypic level, but the molecular mechanisms underlying this effect are not well-understood. In soybean, aphid resistance is conferred by Rag genes. We compared the transcriptional response of four soybean genotypes to aphid feeding to assess how the combination of Rag genes enhanced the soybean resistance to aphid infestation. Results A strong synergistic interaction between Rag1 and Rag2, defined as genes differentially expressed only in the pyramid genotype, was identified. This synergistic effect in the Rag1/2 phenotype was very evident early (6 h after infestation) and involved unique biological processes. However, the response of susceptible and resistant genotypes had a large overlap 12 h after aphid infestation. Transcription factor (TF) analyses identified a network of interacting TF that potentially integrates signaling from Rag1 and Rag2 to produce the unique Rag1/2 response. Pyramiding resulted in rapid induction of phytochemicals production and deposition of lignin to strengthen the secondary cell wall, while repressing photosynthesis. We also identified Glyma.07G063700 as a novel, strong candidate for the Rag1 gene. Conclusions The synergistic interaction between Rag1 and Rag2 in the Rag1/2 genotype can explain its enhanced resistance phenotype. Understanding molecular mechanisms that support enhanced resistance in pyramid genotypes could facilitate more directed approaches for crop improvement.

Methylome and transcriptome analyses of soybean response to bean pyralid larvae

Background Bean pyralid is one of the major leaf-feeding insects that affect soybean crops. DNA methylation can control the networks of gene expressions, and it plays an important role in responses to biotic stress. However, at present the genome-wide DNA methylation profile of the soybean resistance to bean pyralid has not been reported so far. Results Using whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq), we analyzed the highly resistant material (Gantai-2-2, HRK) and highly susceptible material (Wan82–178, HSK), under bean pyralid larvae feeding 0 h and 48 h, to clarify the molecular mechanism of the soybean resistance and explore its insect-resistant genes. We identified 2194, 6872, 39,704 and 40,018 differentially methylated regions (DMRs), as well as 497, 1594, 9596 and 9554 differentially methylated genes (DMGs) in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48 comparisons, respectively. Through the analysis of global methylation and transcription, 265 differentially expressed genes (DEGs) were negatively correlated with DMGs, there were 34, 49, 141 and 116 negatively correlated genes in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48, respectively. The MapMan cluster analysis showed that 114 negatively correlated genes were clustered in 24 pathways, such as protein biosynthesis and modification; primary metabolism; secondary metabolism; cell cycle, cell structure and component; RNA biosynthesis and processing, and so on. Moreover, CRK40; CRK62; STK; MAPK9; L-type lectin-domain containing receptor kinase VIII.2; CesA; CSI1; fimbrin-1; KIN-14B; KIN-14 N; KIN-4A; cytochrome P450 81E8; BEE1; ERF; bHLH25; bHLH79; GATA26, were likely regulatory genes involved in the soybean responses to bean pyralid larvae. Finally, 5 DMRs were further validated that the genome-wide DNA data were reliable through PS-PCR and 5 DEGs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. The results showed an excellent agreement with deep sequencing. Conclusions Genome-wide DNA methylation profile of soybean response to bean pyralid was obtained for the first time. Several specific DMGs which participated in protein kinase, cell and organelle, flavonoid biosynthesis and transcription factor were further identified to be likely associated with soybean response to bean pyralid. Our data will provide better understanding of DNA methylation alteration and their potential role in soybean insect resistance.

Mutations in the Promoter and Coding Regions of Avr3a Cause Gain of Virulence of Phytophthora sojae to Rps3a in Soybean

Phytophthora sojae threatens soybean production worldwide, and the cultivation of soybean cultivars carrying Rps genes is the most effective way to control this pathogen. However, DNA mutations in the Avr genes of P. sojae can escape recognization of the corresponding Rps genes, leading to the loss of soybean resistance. In this study, we investigated sequence polymorphism and transcript level of the Avr3a gene in Chinese isolates of P. sojae. Twenty-four mutations resulting in five unique Avr3a alleles were discovered in the Avr3a coding region from 32 P. sojae isolates. The Avr3a transcripts were detectable in the isolates containing Avr3a(I), Avr3a(II), Avr3a(III), and Avr3a(IV) but not in the isolates containing Avr3a(V). Promoter and 5'-UTR sequence analysis revealed eight unique mutations in the promoter region of Avr3a(V), suggesting that the mutations could result in the loss of Avr3a(V) transcription. Virulence tests indicated the isolates containing Avr3a(II) and Avr3a(IV) were virulent, suggesting that the mutations in the coding regions of Avr3a(II) and Avr3a(IV) caused the gain of virulence to Rps3a. Based on DNA mutations of Avr3a in virulent alleles, two SNP markers and one PCR-based marker were developed successfully for detecting the virulence of P. sojae isolates to Rps3a. These findings provide new insights into escape mechanisms of Avr3a and effective support for accurate pathotype identification of P. sojae using molecular methods.

Soybean cyst nematodes: a destructive threat to soybean production in China

Soybean cyst nematode (SCN), Heterodera glycines, is one of the most important pests in soybean production worldwide. In China, 11 different races of SCN, including a newly identified race ‘X12’ with super-virulence, have been surveyed and found to be distributed in 22 provinces. Among them, races 1, 3 and 4 are dominant in the two principal soybean-producing areas, Northeast China and Huanghuaihai Valley, causing over 120 million dollars of annual yield loss. Rapid and reliable PCR-based approaches have been developed for the molecular diagnosis of SCN. High-throughput methods for the identification of soybean resistance against SCN are also developed with specific single nucleotide polymorphism markers by using Kompetitive Allele Specific PCR technology. Over 10,000 soybean germplasm sources were evaluated for their SCN resistance, and 28 SCN-resistant soybean accessions were selected to construct an applied core collection, which has been used for soybean breeding in China. Recently, the genome sequences of SCN and soybean are publically available, and two major SCN-resistant genes (rhg1 and Rhg4) have been identified in soybean, which greatly facilitate the researches on SCN virulence and soybean resistance, and also soybean resistance breeding against SCN. However, the management of SCN still faces many bottlenecks, for instance, the single resistance genes in soybean cultivars can be easily overcome by new SCN races; the identified resistance genes are inadequate to meet the practical breeding needs; and our understanding of the mechanisms of SCN virulence and soybean resistance to SCN are limited. SCN is a destructive threat to soybean production throughout the world including China. In this review, the major progress on soybean SCN is summarized, mainly focusing on the recent research progress in SCN, soybean resistance to SCN and integrated management of SCN in China, and aiming at a better understanding of the current SCN research status and prospects for future work.

Methylome and Transcriptome Analyses of Soybean Response to Bean Pyralid Larvae

Background: Bean pyralid is an important leaf-feeding insect which affects soybean production. DNA methylation can control the networks of gene expressions, and it plays an important role in the growth, development, and responses to biotic stress. However, the genome-wide DNA methylation profile of the soybean resistance to bean pyralid has not been reported so far. Results: Using whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq), we analysed the highly resistant material (Gantai-2-2) and highly susceptible material (Wan82-178), under the conditions of 0 h and 48 h feeding by bean pyralid larvae, to clarify the molecular mechanism of the soybean resistance and explore its insect-resistant genes. We identified 2,194, 6,872, 39,704, and 40,018 differentially methylated regions (DMRs), as well as 497, 1,594, 9,596, and 9,554 differentially methylated genes (DMGs) in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0, and HSK48/HRK48 comparisons, respectively. We found that 265 differentially expressed genes (DEGs) were negatively correlated with the DMGs, there were 34, 49, 141, and 116 negatively correlated genes in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0, and HSK48/HRK48 comparisons, respectively. The MapMan cluster analysis results indicated that negatively correlated genes in the pathways, such as protein biosynthesis and modification; primary metabolism; secondary metabolism; cell cycle, cell structure and component; RNA biosynthesis and processing, and so on. Finally, the PS-PCR and qRT-PCR were used to validate the expression patterns of several genes and the results showed an excellent agreement with deep sequencing. Conclusions: Through the analysis of global methylation and transcription, we speculated that the expression levels of CRK40; CRK62; STK; L-type lectin-domain containing receptor kinase VIII.2; CesA; CSI1; fimbrin-1; KIN-14B; KIN-14N; KIN-4A; cytochrome P450 81E8; BEE1; ERF; SPATULA; bHLH25; bHLH79; GATA26, were regulated by methylation, and they may potentially play important roles in the soybean responses to bean pyralid larvae. Our results laid a foundation for revealing the occurrence mechanism of soybean response to bean pyralid at the level of DNA methylation.

RESISTANT LEVEL OF SOYBEAN GERMPLASM AGAINST POD SUCKING BUGS (Riptortus spp.)

<p class="abstrakinggris">Increasing productivity of soybean has often been constrained by pod sucking bugs (<em>Riptortus</em> spp.) which caused a serious damage and yield losses up to 80%. Breeding for obtaining soybean variety resistant to pod suckers needs the availability of soybean germplasm resistant to the pest. The study aimed to obtain a candidate for soybean variety resistant to Riptortus spp. through the selection of 100 accessions of soybean. The study included the preparation of test plants and test insects, pest infestations, observations, and looking for a practical screening method for pod sucking pests. The experiment used a completely randomized design for two treatments (infested and non-infested <em>Riptortus</em> spp.). Cikuray variety and PI-092734 accession were used as a control. Results showed that there was a very low correlation among variables observed. Twelve soybean accessions showed a resistance to <em>Riptortus </em>spp., i.e. C7301-113AC-POP, Lokal Madiun-3549, Lokal Klungkung, ML.2974, Singgalang, Lokal Jepara, Lokal Jatim, Lokal Trenggalek, Lokal Tulungagung, Lokal Tabanan, Lokal Blitar, and Lokal Kuningan 10. These accessions were more resistant than the popular released variety such as Wilis, Grobogan, Detam 2, and Gepak Ijo. Small seed size was not a major determinant of soybean resistance to pod suckers. The addition of observational components, i.e. probing preference and oviposition, indicated that crop damage was indirectly influenced by the high frequency of probing and oviposition, although its relation to plant tolerant mechanisms still needs further investigation. Indeterminate plant types require further validation as to whether they contribute significantly to plant resistance against pod sucking insects.</p>