High-Strength Collagen-Based Composite Films Regulated by Water-Soluble Recombinant Spider Silk Proteins and Water Annealing.

Spider silk has attracted extensive attention in the development of high-performance tissue engineering materials because of its excellent physical properties, biocompatibility, and biodegradability. Although high-molecular-weight recombinant spider silk proteins can be obtained through metabolic engineering of host bacteria, the solubility of the recombinant protein products is always poor. Strong denaturants and organic solvents have thus had to be exploited for their dissolution, and this seriously limits the applications of recombinant spider silk protein-based composite biomaterials. Herein, through adjusting the temperature, ionic strength, and denaturation time during the refolding process, we successfully prepared water-soluble recombinant spider major ampullate spidroin 1 (sMaSp1) with different repeat modules (24mer, 48mer, 72mer, and 96mer). Then, MaSp1 was introduced into the collagen matrix for fabricating MaSp1-collagen composite films. The introduction of spider silk proteins was demonstrated to clearly alter the internal structure of the composite films and improve the mechanical properties of the collagen-based films and turn the opaque protein films into transparency ones. More interestingly, the composite film prepared with sMaSp1 exhibited better performance in mechanical strength and cell adhesion compared to that prepared with water-insoluble MaSp1 (pMaSp1), which might be attributed to the effect of the initial dissolved state of MaSp1 on the microstructure of composite films. Additionally, the molecular weight of MaSp1 was also shown to significantly influence the mechanical strength (enhanced to 1.1- to 2.3-fold) and cell adhesion of composite films, and 72mer of sMaSp1 showed the best physical properties with good bioactivity. This study provides a method to produce recombinant spider silk protein with excellent water solubility, making it possible to utilize this protein under environmentally benign, mild conditions. This paves the way for the application of recombinant spider silk proteins in the development of diverse composite biomaterials.

Transgenic RXLR Effector PITG_15718.2 Suppresses Immunity and Reduces Vegetative Growth in Potato.

Phytophthora infestans causes the severe late blight disease of potato. During its infection process, P. infestans delivers hundreds of RXLR (Arg-x-Leu-Arg, x behalf of any one amino acid) effectors to manipulate processes in its hosts, creating a suitable environment for invasion and proliferation. Several effectors interact with host proteins to suppress host immunity and inhibit plant growth. However, little is known about how P. infestans regulates the host transcriptome. Here, we identified an RXLR effector, PITG_15718.2, which is upregulated and maintains a high expression level throughout the infection. Stable transgenic potato (Solanum tuberosum) lines expressing PITG_15718.2 show enhanced leaf colonization by P. infestans and reduced vegetative growth. We further investigated the transcriptional changes between three PITG_15718.2 transgenic lines and the wild type Désirée by using RNA sequencing (RNA-Seq). Compared with Désirée, 190 differentially expressed genes (DEGs) were identified, including 158 upregulated genes and 32 downregulated genes in PITG_15718.2 transgenic lines. Eight upregulated and nine downregulated DEGs were validated by real-time RT-PCR, which showed a high correlation with the expression level identified by RNA-Seq. These DEGs will help to explore the mechanism of PITG_15718.2-mediated immunity and growth inhibition in the future.