Involvement of 2'-5'oligoadenylate synthetase-like proteinin the survivalof Mycobacterium tuberculosis avirulent strain in macrophages

Mycobacterium tuberculosis(M.tuberculosis)can replicate in the macrophage by interfering with many host protein functions.While it is far from known these host proteins for controlling M.tuberculosis infection.Herein,we infected macrophages including THP-1 and Raw264.7 cells with M.tuberculosis and identified the differentially expressed genes(DEGs)in the interferon signaling pathway.Among them,2'-5'oligoadenylate synthetase-like(OASL)underwent the greatest upregulation in M.tuberculosis-infected macrophages.Knockdown of the expression of OASL attenuated M.tuberculosis survival in macrophages.Further,bioinformatics analysis revealed the potential interaction axis of OASL-TAB3-RvO127,which was further validated by the yeast-two-hybrid(Y2H)assay and Co-IP.This interaction axis might regulate the M.tuberculosis survival and proliferation in macrophages.The study reveals a possible role of OASL during M.tuberculosis infection as a target to control its propagation.

A splice site mutation in the FvePHP gene is associated with leaf development and f lowering time in woodland strawberry

Leaves and f lowers are crucial for the growth and development of higher plants.In this study we identified a mutant with narrow leaf lets and early f lowering(nlef)in an ethyl methanesulfonate-mutagenized population of woodland strawberry(Fragaria vesca)and aimed to identify the candidate gene.Genetic analysis revealed that a single recessive gene,nlef,controlled the mutant phenotype.We found that FvH4_1g25470,which encodes a putative DNA polymeraseαwith a polymerase and histidinol phosphatase domain(PHP),might be the candidate gene,using bulked segregant analysis with whole-genome sequencing,molecular markers,and cloning analyses.A splice donor site mutation(C to T)at the 5-end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants.FvePHP was localized in the nucleus and was highly expressed in leaves.Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves.Overexpression of the FvePHP gene can largely restore the mutant phenotype.The expression levels of FveSEP1,FveSEP3,FveAP1,FveFUL,and FveFT were higher in the mutants than those in‘Yellow Wonder’plants,probably contributing to the early f lowering phenotype in mutant plants.Our results indicate that mutation in FvePHP is associated with multiple developmental pathways.These results aid in understanding the role of DNA polymerase in strawberry development.

Woodland strawberry WRKY71 acts as a promoter of flowering via a transcriptional regulatory cascade

The WRKY proteins are a large family of transcription factors that play important roles in stress responses and plant development.However,the roles of most WRKYs in strawberry are not well known.In this study,FvWRKY71 was isolated from the woodland strawberry‘Ruegen’.FvWRKY71 was highly expressed in the shoot apex and red fruit.Subcellular localization analysis showed that FvWRKY71 was located in the nucleus.Transactivation analysis showed that FvWRKY71 presented transcriptional activation activity in yeast.Overexpression of FvWRKY71 in Arabidopsis and woodland strawberry revealed early flowering in the transgenic plants compared with the wild-type control.Gene expression analysis indicated that the transcript levels of the flowering time and development integrator genes AP1,LFY,FT,AGL42,FUL,FPF1,SEP1,SEP2,and SEP3 were increased in FvWRKY71-overexpressing Arabidopsis and strawberry plants compared with the wild-type controls,which may result in accelerated flowering in transgenic plants.Furthermore,FvWRKY71 was proven to directly bind to the W-boxes(TTGACT/C)of the FvFUL,FvSEP1,FvAGL42,FvLFY,and FvFPF1 promoters in vitro and in vivo.Taken together,our results reveal a transcriptional regulatory cascade of FvWRKY71 involved in promoting flowering in woodland strawberry.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genomewide RNA Interference Screening

Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single-and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.