Beet cyst nematode (Heterodera schachtii Schm.) is one of the most damaging pests in sugar beet growing areas around the world. The Hs1pro-1 and cZR3 genes confer resistance to the beet cyst nematode, and both were cloned from sugar beet translocation line (A906001). The translocation line carried the locus from B. procumbens chromosome 1 including Hs1pro-1 gene and resistance gene analogs (RGA), which confer resistance to Heterodera schachtii. In this research, BvHs1pro-1 and BvcZR3 genes were transferred into oilseed rape to obtain different transgenic lines by A. tumefaciens mediated transformation method. The cZR3Hs1pro-1 gene was pyramided into the same plants by crossing homozygous cZR3 and Hs1pro-1 plants to identify the function and interaction of cZR3 and Hs1pro-1 genes. In vitro and in vivo cyst nematode resistance tests showed that cZR3 and Hs1pro-1 plants could be infested by beet cyst nematode (BCN) juveniles, however a large fraction of penetrated nematode juveniles was not able to develop normally and stagnated in roots of transgenic plants, consequently resulting in a significant reduction in the number of developed nematode females. A higher efficiency in inhibition of nematode females was observed in plants expressing pyramiding genes than in those only expressing a single gene. Molecular analysis demonstrated that BvHs1pro-1 and BvcZR3 gene expressions in oilseed rape constitutively activated transcription of plant-defense related genes such as NPR1 (non-expresser of PR1), SGT1b (enhanced disease resistance 1) and RAR1 (suppressor of the G2 allele of skp1). Transcript of NPR1 gene in transgenic cZR3 and Hs1pro-1 plants were slightly up-regulated, while its expression was considerably enhanced in cZR3Hs1pro-1 gene pyramiding plants. The expression of EDS1 gene did not change significantly among transgenic cZR3, Hs1pro-1 and cZR3Hs1pro-1 gene pyramiding plants and wild type. The expression of SGT1b gene was slightly up-regulated in transgenic cZR3 and Hs1pro-1 plants compared with the wild type, however, its expression was not changed in cZR3Hs1pro-1 gene pyramiding plant and had no interaction effect. RAR1 gene expression was significantly up-regulated in transgenic cZR3 and cZR3Hs1pro-1 genes pyramiding plants, but almost no expression was found in Hs1pro-1 transgenic plants. These results show that nematode resistance genes from sugar beet were functional in oilseed rape and conferred BCN resistance by activation of a CC-NBS-LRR R gene mediated resistance response. The gene pyramiding had enhanced resistance, thus offering a novel approach for the BCN control by preventing the propagation of BCN in oilseed rape. The transgenic oilseed rape could be used as a trap crop to offer an alternative method for beet cyst nematode control.

Drought stress is a common issue that affects agriculture production. The soybean plant is one of the drought-sensitive crops that could lose the yield of up to 40% under severe drought years. Improvements in drought tolerance have been one of the main objectives of breeding programs in soybean. In this study, 62 soybeans [Glycine max (L.) Merri.] germplasms including landraces and elite cultivars were evaluated by analyzing canopy and root morphological characteristics at seedling stage under polyethylene glycol (2.5 M PEG 8000, − 0.54 MPa) simulated drought stress. The results showed that the wilting index of sixty-two soybean germplasms showed significant polymorphism differences after 4 days PEG treatment. The lowest wilting index was 2.75 in Tiefeng 31, while the largest reached 9 in Fengdou 93 and Songzidou. The wilting index was positively correlated with plant height, electrolyte leakage, and negatively correlated with a dry weight of the above-ground part, stomatal conductance, and transpiration rate by regression analysis. The root system architecture (RSA) study showed root-canopy ratio, root length, number of the lateral roots, root surface area, and root volume were significantly negatively correlated with the wilting index and positively correlated with the root volume. Based on these results, sixty-two soybean germplasms were comprehensively evaluated for drought-tolerant cultivar by the principal component analysis (PCA). The results showed that Tiefeng 31 was the most drought-tolerant elite cultivar. However, Fengdou 93 was the most droughtsensitive elite variety. The drought tolerance screening results were consistent with the wilting index and RSA analysis, especially the root length. Both droughts tolerant and sensitive elite cultivar could be further used to breed drought-tolerant germplasms and to clarify the drought tolerance mechanism in soybean.

The HD-Zip transcription factors play a crucial role in plant development, secondary metabolism, and abiotic stress responses, but little is known about HD-Zip I genes in soybean. Here, a homeodomain-leucine zipper gene designated GmHdz4 was isolated. Chimeric soybean plants, GmHdz4 overexpressing (GmHdz4-oe), and gene-editing via CRISPR/Cas9 (gmhdz4) in hairy roots, were generated to examine the GmHdz4 gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed GmHdz4 belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as AtHB12, Oshox12, and Gshdz4. The GmHdz4 was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that GmHdz4 expression varied in tissues and was induced by PEG-simulated drought stress. The gmhdz4 showed promoted growth of aboveground parts, and its root system architecture, including the total root length, the root superficial area, and the number of root tips were significantly higher than those of GmHdz4-oe even the non-transgenic line (NT) on root tips number. The better maintenance of turgor pressure by osmolyte accumulation, and the higher activity of antioxidant enzymes to scavenge reactive oxygen species, ultimately suppressed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2−), and malondialdehyde (MDA), conferring higher drought tolerance in gmhdz4 compared with both GmHdz4-oe and NT. Together, our results provide new insights for future research on the mechanisms by which GmHdz4 gene-editing via CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.

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Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) causes an estimated economic loss of about USD 3 billion each year in soybean (Glycine max L.) production worldwide. Overexpression of resistance genes against SCN provides a powerful approach to develop SCN resistance cultivars in soybean. The clarification of molecular characterization in transformation events is a prerequisite for ecological risk assessment, food safety, and commercial release of genetically modified crops. Here, we generated transgenic events harboring the BCN (beet cyst nematode) resistance Hs1pro−1 gene using the Agrobacterium-mediated method in soybean, evaluated their resistance to SCN infection, and clarified the molecular characterization of one of the transformation events. Five independent and stable inheritable transformation events were generated by an Agrobacterium-mediated transformation method. SCN resistance tests showed the average number of developed females per plant and female index (FI) in T4 ZHs1-1, ZHs1-2, ZHs1-3, ZHs1-4, and ZHs1-5 transformation events were significantly lower than that in the nontransgenic control. Among these, the ZHs1-2 transformation event had the lowest number of developed females per plant and FI. Southern hybridization showed the exogenous target Hs1pro−1 gene was inserted in one copy and the Bar gene was inserted two copies in the ZHs1-2 transformation event. The exogenous T-DNA fragment was integrated in the reverse position of Chr02: 5351566–5231578 (mainly the Bar gene expression cassette) and in the forward position of Chr03: 17083358–17083400 (intact T-DNA, including Hs1pro−1and Bar gene expression cassette) using a whole genome sequencing method (WGS). The results of WGS method and Southern hybridization were consistent. All the functional elements of exogenous T-DNA fragments were verified by PCR using specific primer pairs in the T5 and T6 ZHs1-2 transformation events. These results demonstrated that the overexpression of Hs1pro−1 gene enhanced SCN resistance, and provide an important reference for the biosafety assessment and the labeling detection in transformation event ZHs1-2.