Generation and Identification of the Number of Copies of Exogenous Genes and the T-DNA Insertion Site in SCN-Resistance Transformation Event ZHs1-2.

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-1 and 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.

BvcZR3 and BvHs1pro-1 Genes Pyramiding Enhanced Beet Cyst Nematode (Heterodera schachtii Schm.) Resistance in Oilseed Rape (Brassica napus L.).

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.

Physiological Regulation of Photosynthetic-Related Indices, Antioxidant Defense, and Proline Anabolism on Drought Tolerance of Wild Soybean (Glycine soja L.).

Wild soybean (Glycine soja L.), drought-tolerant cultivar Tiefeng 31 (Glycine max L.), and drought-sensitive cultivar Fendou 93 (Glycine max L.) were used as materials to investigate the drought tolerance mechanism after 72 h 2.5 M PEG 8000 (osmotic potential -0.54 MPa)-simulated drought stress at the seedling stage. The results indicated that the leaves of the G. soja did not wilt under drought stress. However, both the drought-tolerant and drought-sensitive cultivated soybean cultivars experienced varying degrees of leaf wilt. Notably, the drought-sensitive cultivated soybean cultivars exhibited severe leaf wilt after the drought stress. Drought stress was determined to have a significant impact on the dry matter of the above-ground part of the drought-sensitive cultivar Fendou 93, followed by the drought-tolerant cultivar Tiefeng 31, with the lowest reduction observed in G. soja. Furthermore, the presence of drought stress resulted in the closure of leaf stomata. G. soja exhibited the highest proportion of stomatal opening per unit area, followed by the drought-tolerant cultivar Tiefeng 31, while the drought-sensitive cultivar Fendou 93 displayed the lowest percentage. Photosynthesis-related indexes, including photosynthetic rate, intercellular CO2, transpiration rate, and stomatal conductance, decreased in Fendou 93 and Tiefeng 31 after drought stress, but increased in G. soja. In terms of the antioxidant scavenging system, lower accumulation of malondialdehyde (MDA) was observed in G. soja and Tiefeng 31, along with higher activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) to counteract excess reactive oxygen species and maintain cell membrane integrity. In contrast, the drought-sensitive cultivar Fendou 93 had higher MDA content and higher activities of ascorbate peroxidase (APX, EC 1.11.1.11) and peroxidase (POD, 1.11.1.7). G. soja and Tiefeng 31 also exhibited less accumulation of osmolytes, including soluble sugar, soluble protein, and free proline content. The activities of δ-OAT, ProDH, and P5CS, key enzymes in proline anabolism, showed an initial increase under drought stress, followed by a decrease, and then an increase again at the end of drought stress in G. soja. Before drought stress, Tiefeng 31 had higher activities of ProDH and P5CS, which decreased with prolonged drought stress. Fendou 93 experienced an increase in the activities of δ-OAT, ProDH, and P5CS under drought stress. The δ-OAT gene expression levels were up-regulated in all three germplasms. The expression levels of the P5CS gene in Fendou 93 and Tiefeng 31 were down-regulated, while G. soja showed no significant change. The expression of the P5CR gene and ProDH gene was down-regulated in Fendou 93 and Tiefeng 31, but up-regulated in G. soja. This indicates that proline content is regulated at both the transcription and translation levels.

CRISPR/Cas9 mediated gene-editing of GmHdz4 transcription factor enhances drought tolerance in soybean (Glycine max [L.] Merr.).

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.