Table_1_Overexpression of Cinnamoyl-CoA Reductase 2 in Brassica napus Increases Resistance to Sclerotinia sclerotiorum by Affecting Lignin Biosynthesis.DOCX

Sclerotinia sclerotiorum causes severe yield and economic losses for many crop and vegetable species, especially Brassica napus. To date, no immune B. napus germplasm has been identified, giving rise to a major challenge in the breeding of Sclerotinia resistance. In the present study, we found that, compared with a Sclerotinia-susceptible line (J902), a Sclerotinia-resistant line (J964) exhibited better xylem development and a higher lignin content in the stems, which may limit the invasion and spread of S. sclerotiorum during the early infection period. In addition, genes involved in lignin biosynthesis were induced under S. sclerotiorum infection in both lines, indicating that lignin was deposited proactively in infected tissues. We then overexpressed BnaC.CCR2.b, which encodes the first rate-limiting enzyme (cinnamoyl-CoA reductase) that catalyzes the reaction of lignin-specific pathways, and found that overexpression of BnaC.CCR2.b increased the lignin content in the stems of B. napus by 2.28–2.76% under normal growth conditions. We further evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines at the flower-termination stage and found that the disease lesions on the stems of plants in the T2 and T3 generations decreased by 12.2–33.7% and 32.5–37.3% compared to non-transgenic control plants, respectively, at 7days post-inoculation (dpi). The above results indicate that overexpression of BnaC.CCR2.b leads to an increase in lignin content in the stems, which subsequently leads to increased resistance to S. sclerotiorum. Our findings demonstrate that increasing the lignin content in the stems of B. napus is an important strategy for controlling Sclerotinia.

核盘菌（Sclerotinia sclerotiorum）可对多种农作物及蔬菜物种造成严重的产量与经济损失，其中对甘蓝型油菜（Brassica napus）的危害尤为突出。迄今为止尚未发现免疫型甘蓝型油菜种质资源，这给核盘菌抗性育种带来了重大挑战。本研究发现，相较于核盘菌感病系J902，核盘菌抗病系J964的茎部木质部发育更为优良，茎内木质素（lignin）含量更高，这或许可在侵染早期限制核盘菌的入侵与扩散。此外，两个品系中参与木质素生物合成的基因均在核盘菌侵染条件下被诱导表达，提示木质素会主动沉积于受侵染的组织中。我们随后对BnaC.CCR2.b进行了过表达，该基因编码催化木质素特异性合成通路反应的首个限速酶——肉桂酰辅酶A还原酶（cinnamoyl-CoA reductase）。实验结果显示，在正常生长条件下，BnaC.CCR2.b过表达可使甘蓝型油菜茎部的木质素含量提升2.28%~2.76%。我们进一步在终花期对BnaC.CCR2.b过表达株系的核盘菌抗性进行了评价，结果发现，在接种后7天（days post-inoculation，dpi），T2和T3代转基因株系的茎部病斑面积分别较非转基因对照植株降低12.2%~33.7%与32.5%~37.3%。上述结果表明，过表达BnaC.CCR2.b可提升甘蓝型油菜茎部的木质素含量，进而增强其对核盘菌的抗性。本研究证实，提高甘蓝型油菜茎部木质素含量是防控核盘菌病害的重要策略。