Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 μM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 μM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

Background: Wild apple, Malus sieversii, is an endangered species and a valuable genetic resource that requires a variety of conservation techniques. This study aimed to investigate the influence of different concentrations of hormones on wild apple regeneration from leaf and stem explants to establish an optimal regeneration system. Results: Leaves and stems derived from seedlings were cultured on several media supplemented with various concentrations of thidiazuron (TDZ) or 6-benzylaminopurine (BA) in different combinations with 1-naphthaleneacetic acid (NAA). The results showed that the most efficient shoot formation media (35% and 90%) were MS medium containing 4.0 mg L⁻¹ TDZ and 1.0 mg L⁻¹ NAA for leaf explants and MS medium containing 1.0 mg L⁻¹ BA without NAA for stem explant. MS medium supplemented with 0.4 mg L⁻¹ BA and 0.1 mg L⁻¹ NAA (for shoot multiplication) and 1/2 MS + 0.1 mg L⁻¹ NAA + 1.5% sucrose (for rooting) were effective media. Shoot regeneration from leaf explants was the most effective when the explants were placed abaxial side down onto the medium and were subjected to a pre-treatment of 3 weeks in darkness. Conclusions: An optimized regeneration system for M. sieversii that allowed regeneration within 2–3 months developed. The protocol developed herein can be used in large-scale clonal propagation for the conservation of wild apple, M. sieversii.

Huanglongbing (HLB) is a highly detrimental citrus disease associated with ‘Candidatus Liberibacter asiaticus’, a nonculturable alpha-proteobacterium. Characterization of the bacterial populations is important for development of disease management strategies. In this study, the ‘Ca. L. asiaticus’ populations in eight provinces in southern China where HLB is endemic were analyzed based on tandem repeat number (TRN) variations in a previously characterized genomic locus CLIBASIA_01645. Of the 224 HLB samples collected, 175 (78.3%) samples yielded single polymerase chain reaction (PCR) amplicons (the single amplicon group, SAG) and 49 (21.7%) samples produced multiple PCR amplicons (the multiple amplicon group, MAG). Variations in SAG are summarized by Nei's diversity index (H) and ratio of TRN ≤ 10/TRN > 10 genotypes (R₁₀). Variations in the MAG are described by the percentage of occurrence (P_MAG). At an orchard-level comparison, the ‘Ca. L. asiaticus’ population from a Guangdong orchard (n = 24) showed H = 0.50, R₁₀ = 23, and P_MAG = 0, significantly different from that of the non-Guangdong orchards in Yunnan (n = 23), H = 0.83, R₁₀ = 2.3, and P_MAG = 11.5, and in Hainan (n = 35), H = 0.88, R₁₀ = 1.5, and P_MAG = 16.7. In a region-level consideration, the Guangdong ‘Ca. L. asiaticus’ population (n = 78) was H = 0.77, R₁₀ = 25, and P_MAG = 1.3, whereas the non-Guangdong population (n = 84) was H = 0.91, R₁₀ = 1.6, and P_MAG = 26.9. Overall, significant differences were observed between the ‘Ca. L. asiaticus’ population from Guangdong Province and those from the other provinces. A strong aggregation of TRN = 6, 7, and 8 genotypes is characteristic to the ‘Ca. L. asiaticus’ population in Guangdong. Referenced to genome annotation, we propose that rearrangement of tandem repeats at locus CLIBASIA_01645 could be associated with bacterial environmental adaptation.

Gene expression profiles are powerful tools for investigating mechanisms of plant stress tolerance. Betula platyphylla (birch) is a widely distributed tree, but its drought-tolerance mechanism has been little studied. Using RNA-Seq, we identified 2917 birch genes involved in its response to drought stress. These drought-responsive genes include the late embryogenesis abundant (LEA) family, heat shock protein (HSP) family, water shortage-related and ROS-scavenging proteins, and many transcription factors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF) and myeloblastosis oncogene (MYB) families were the most abundant. BpERF2 and BpMYB102, which were strongly induced by drought and had high transcription levels, were selected to study their regulatory networks. BpERF2 and BpMYB102 both played roles in enhancing drought tolerance in birch. Chromatin immunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genes such as those in the LEA and HSP families, while BpMYB102 regulated genes such as Pathogenesis-related Protein 1 (PRP1) and 4-Coumarate:Coenzyme A Ligase 10 (4CL10). Multiple genes were regulated by both BpERF2 and BpMYB102. We further characterized the function of some of these genes, and the genes that encode Root Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs were found to be involved in drought tolerance. Therefore, our results suggest that BpERF2 and BpMYB102 serve as transcription factors that regulate a series of drought-tolerance genes in B. platyphylla to improve drought tolerance.

DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. In the present study, we developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are then identified using mass spectrometry. Using R-ChIP, we identified 439 proteins that potentially bind to the promoter of the Arabidopsis thaliana gene AtCAT3 (AT1G20620). According to functional annotation, we randomly selected 5 transcription factors from these candidates, including bZIP1664, TEM1, bHLH106, BTF3, and HAT1, to verify whether they in fact bind to the AtCAT3 promoter. The binding of these 5 transcription factors was confirmed using chromatin immunoprecipitation quantitative real-time PCR and electrophoretic mobility shift assays. In addition, we improved the R-ChIP method using plants in which the DNA of interest had been transiently introduced, which does not require the T-DNA integration, and showed that this substantially improved the protein capture efficiency. These results together demonstrate that R-ChIP has a wide application to characterize chromatin composition and isolate upstream regulators of a specific gene.