CUBIC: A Versatile Cumate-Based Inducible CRISPRi System in <i>Streptomyces</i>

Streptomyces, a genus of Gram-positive bacteria, is known as nature’s medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of Streptomyces. We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in Streptomyces. Its broad application is shown by the specific and nondisruptive knockdown of genes involved in growth, development and antibiotic production in various Streptomyces species. To facilitate hyper-efficient plasmid construction, we adapted the Golden Gate assembly to achieve 100% cloning efficiency of the protospacers. We expect that the versatile plug-and-play CUBIC system will create new opportunities for research and innovation in the field of Streptomyces

Overexpression of the maize transcription factor ZmVQ52 accelerates leaf senescence in Arabidopsis.

Leaf senescence plays an important role in the improvement of maize kernel yields. However, the underlying regulatory mechanisms of leaf senescence in maize are largely unknown. We isolated ZmVQ52 and studied the function of ZmVQ52 which encoded, a VQ family transcription factor. ZmVQ52 is constitutively expressed in maize tissues, and mainly expressed in the leaf; it is located in the nucleus of maize protoplasts. Four WRKY family proteins-ZmWRKY20, ZmWRKY36, ZmWRKY50, and ZmWRKY71-were identified as interacting with ZmVQ52. The overexpression of ZmVQ52 in Arabidopsis accelerated premature leaf senescence. The leaf of the ZmVQ52-overexpression line showed a lower chlorophyll content and higher senescence rate than the WT. A number of leaf senescence regulating genes were up-regulated in the ZmVQ52-overexpression line. Additionally, hormone treatments revealed that the leaf of the ZmVQ52-overexpressed line was more sensitive to salicylic acid (SA) and jasmonic acid (JA), and had an enhanced tolerance to abscisic acid (ABA). Moreover, a transcriptome analysis of the ZmVQ52-overexpression line revealed that ZmVQ52 is mainly involved in the circadian pathway and photosynthetic pathways

Dynamics of germination stimulants dehydrocostus lactone and costunolide in the root exudates and extracts of sunflower

Orobanche cumana Wallr. (Orobanche cernua Loefl.) causes severe yield losses of confectionary sunflower in China. While germination of O. cumana is stimulated by sesquiterpene lactones (STLs) from host sunflower (Helianthus annuus L.). Dehydrocostus lactone and costunolide isolated from sunflower root exudates are known as STLs to specifically induce O. cumana germination. Two major confectionary sunflower cultivars, SH363 (highly susceptible to O. cumana) and TH33 (resistant to O. cumana), were planted in China. However, STLs in these two sunflower cultivars has remained unknown. To identify STLs from root and exudates of sunflower for better understanding the role of stimulants in parasitic interaction of sunflower and O. cumana, we tested dehydrocostus lactone (DCL) and costunolide (CL) in root and root exudates of susceptible and resistant sunflower cultivars. The stimulant activity of sunflower root exudate and root extract to germination of O. cumana were also determined. Dehydrocostus lactone and costunolide were identified through ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Both DCL and CL were found in root extracts and root exudates in the whole tested time point from two sunflower cultivars. The concentration of dehydrocostus lactone was higher than that of costunolide at the same tested growth stage of each sunflower cultivar. It was observed that higher quantity of dehydrocostus lactone in susceptible cultivar than resistant cultivar of root and root exudates at later tested developmental stages. However, the amount of CL was no significant difference between SH363 and TH33 at all tested stages. The release amount of DCL from susceptible cultivar is 3.7 folds that of resistant cultivar at 28 DAT. These findings suggested that DCL was the one of the major signal compound in these two sunflower cultivars, and lower dehydrocostus lactone might contribute to the resistance of sunflower TH33 to O. cumana