CPR5-mediated nucleo-cytoplasmic localization of IAA12 and IAA19 controls lateral root development during abiotic stress.

Plasticity of the root system architecture (RSA) is essential in enabling plants to cope with various environmental stresses and is mainly controlled by the phytohormone auxin. Lateral root development is a major determinant of RSA. Abiotic stresses reduce auxin signaling output, inhibiting lateral root development; however, how abiotic stress translates into a lower auxin signaling output is not fully understood. Here, we show that the nucleo-cytoplasmic distribution of the negative regulators of auxin signaling AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE 12 (AUX/IAA12 or IAA12) and IAA19 determines lateral root development under various abiotic stress conditions. The cytoplasmic localization of IAA12 and IAA19 in the root elongation zone enforces auxin signaling output, allowing lateral root development. Among components of the nuclear pore complex, we show that CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES 5 (CPR5) selectively mediates the cytoplasmic translocation of IAA12/19. Under abiotic stress conditions, CPR5 expression is strongly decreased, resulting in the accumulation of nucleus-localized IAA12/19 in the root elongation zone and the suppression of lateral root development, which is reiterated in the cpr5 mutant. This study reveals a regulatory mechanism for auxin signaling whereby the spatial distribution of AUX/IAA regulators is critical for lateral root development, especially in fluctuating environmental conditions.

JULGI-mediated increment in phloem transport capacity relates to fruit yield in tomato.

The continuous growth of the global population and the increase in the amount of arid land has severely constrained agricultural crop production. To solve this problem, many researchers have attempted to increase productivity through the efficient distribution of energy; however, the direct relationship between the plant vasculature, specifically phloem development, and crop yield is not well established. Here, we demonstrate that an optimum increase in phloem-transportation capacity by reducing SIJUL expression leads to improved sink strength in tomato (Solanum lycopersicum L.). SIJUL, a negative regulator of phloem development, suppresses the translation of a positive regulator of phloem development, SlSMXL5. The suppression of SlJUL increases the number of phloem cells and sucrose transport, but only an optimal reduction of SlJUL function greatly enhances sink strength in tomato, improving fruit setting, and yield contents by 37% and 60%, respectively. We show that the increment in phloem cell number confers spare transport capacity. Our results suggest that the control of phloem-transport capacity within the threshold could enhance the commitment of photosynthates to instigate yield improvement.

HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis.

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on class I interfering repair, with a minority formed via the class II pathway. Class II repair is limited by anti-recombination pathways; however, similar pathways repressing class I crossovers have not been identified. Here, we performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and class I proteins, including HEI10, PTD, MSH5 and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Emerging roles of RNA-binding proteins in plant development.

RNA-binding proteins (RBPs) influence the fate of target RNAs via direct interactions. During transcription, RBPs and interacting partners are recruited to and modify transcripts, after which they may also participate in critical steps to generate functional RNA. RBP-RNA interactions govern post-transcriptional processing of RNA, consequently regulating gene expression in a spatio-temporal manner. In plants, an increasing number of proteins have been classified as RBPs, many of which have been shown to function as key players in diverse developmental processes. However, a comprehensive understanding of how RBPs function, which RNAs are targeted, and where RBP-RNA interactions occur within plant cells is lacking. Here, we discuss recent findings in the field and newly defined roles for RBPs in plant growth and development. We also describe the mechanistic effects of RBPs on target RNA metabolism and translation.

In vivo action of RNA G-quadruplex in phloem development.

Phloem network integrates cellular energy status into post-embryonic growth, and development by tight regulation of carbon allocation. Phloem development involves complicated coordination of cell fate determination, cell division, and terminal differentiation into sieve elements (SEs), functional conduit. All of these processes must be tightly coordinated, for optimization of systemic connection between source supplies and sink demands throughout plant life cycle, that has substantial impact on crop productivity. Despite its pivotal role, surprisingly, regulatory mechanisms underlying phloem development have just begun to be explored, and we recently identified a novel translational regulatory network involving RNA G-quadruplex and a zinc-finger protein, JULGI, for phloem development. From this perspective, we further discuss the role of RNA G-quadruplex on post-transcriptional control of phloem regulators, as a potential interface integrating spatial information for asymmetric cell division, and phloem development. [BMB Reports 2018; 51(11): 547-548].

Translational control of phloem development by RNA G-quadruplex-JULGI determines plant sink strength.

The emergence of a plant vascular system was a prerequisite for the colonization of land; however, it is unclear how the photosynthate transporting system was established during plant evolution. Here, we identify a novel translational regulatory module for phloem development involving the zinc-finger protein JULGI (JUL) and its targets, the 5' untranslated regions (UTRs) of the SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs, which is exclusively conserved in vascular plants. JUL directly binds and induces an RNA G-quadruplex in the 5' UTR of SMXL4/5, which are key promoters of phloem differentiation. We show that RNA G-quadruplex formation suppresses SMXL4/5 translation and restricts phloem differentiation. In turn, JUL deficiency promotes phloem formation and strikingly increases sink strength per seed. We propose that the translational regulation by the JUL/5' UTR G-quadruplex module is a major determinant of phloem establishment, thereby determining carbon allocation to sink tissues, and that this mechanism was a key invention during the emergence of vascular plants.

Nucleosomes and DNA methylation shape meiotic DSB frequency in Arabidopsis thaliana transposons and gene regulatory regions.

Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis thaliana SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within A. thaliana genes and transposons, with significance for the diversity and evolution of plant genomes.

Red wines and flavonoids diminish Staphylococcus aureus virulence with anti-biofilm and anti-hemolytic activities.

The emergence of antibiotic resistant Staphylococcus aureus presents a worldwide problem that requires non-antibiotic strategies. This study investigated the anti-biofilm and anti-hemolytic activities of four red wines and two white wines against three S. aureus strains. All red wines at 0.5-2% significantly inhibited S. aureus biofilm formation and hemolysis by S. aureus, whereas the two white wines had no effect. Furthermore, at these concentrations, red wines did not affect bacterial growth. Analyses of hemolysis and active component identification in red wines revealed that the anti-biofilm compounds and anti-hemolytic compounds largely responsible were tannic acid, trans-resveratrol, and several flavonoids. In addition, red wines attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is killed by S. aureus. These findings show that red wines and their compounds warrant further attention in antivirulence strategies against persistent S. aureus infection.

Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7.

E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50µg/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shiga-like toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection.

Thermoresponsive oligomers reduce Escherichia coli O157:H7 biofouling and virulence.

Thermoresponsive polymers have potential biomedical applications for drug delivery and tissue engineering. Here, two thermoresponsive oligomers were synthesized, viz. oligo(N-isopropylacrylamide) (ONIPAM) and oligo(N-vinylcaprolactam) (OVCL), and their anti-biofouling abilities investigated against enterohemorrhagic E. coli O157:H7, which produces Shiga-like toxins and forms biofilms. Biofilm formation (biofouling) is closely related to E. coli O157:H7 infection and constitutes a major mechanism of antimicrobial resistance. The synthetic OVCL (MW 679) and three commercial OVCLs (up to MW 54,000) at 30 µg ml(-1) were found to inhibit biofouling by E. coli O157:H7 at 37 °C by more than 80% without adversely affecting bacterial growth. The anti-biofouling activity of ONIPAM was weaker than that of OVCL. However, at 25 °C, ONIPAM and OVCL did not affect E. coli O157:H7 biofouling. Transcriptional analysis showed that OVCL temperature-dependently downregulated curli genes in E. coli O157:H7, and this finding was in line with observed reductions in fimbriae production and biofouling. In addition, OVCL downregulated the Shiga-like toxin genes stx1 and stx2 in E. coli O157:H7 and attenuated its in vivo virulence in the nematode Caenorhabditis elegans. These results suggest that OVCL has potential use in antivirulence strategies against persistent E. coli O157:H7 infection.

Diverse plant extracts and trans-resveratrol inhibit biofilm formation and swarming of Escherichia coli O157:H7.

Infection with enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem. Of the 498 plant extracts screened against EHEC, 16 inhibited the formation of biofilm of EHEC by >85% without inhibiting the growth of planktonic cells, and 14 plant extracts reduced the swarming motility of EHEC. The most active extract, Carex dimorpholepis, decreased swimming and swarming motilities and curli formation. Transcriptional analyses showed that the extract of C. dimorpholepis repressed curli genes, various motility genes, and AI-2 quorum sensing genes, which was corroborated by reduction in the production of fimbria, motility, and biofilm by EHEC. Trans-resveratrol at 10 µg ml(-1) in the extract of C. dimorpholepis was found to be a new anti-biofilm compound against EHEC, but importantly, the extract of C. dimorpholepis and trans-resveratrol did not inhibit the fomation of biofilm in four commensal E. coli strains. Furthermore, the extract of C. dimorpholepis decreased the adhesion of EHEC cells to human epithelial cells without affecting the viability of these cells.

Inhibition of Pseudomonas aeruginosa and Escherichia coli O157:H7 biofilm formation by plant metabolite ε-viniferin.

Pathogenic biofilms are associated with persistent infection due to their high resistances to diverse antibiotics. Pseudomonas aeruginosa infects plants, animals, and humans and is a major cause of nosocomial diseases in patients with cystic fibrosis. In the present study, the antibiofilm abilities of 522 plant extracts against P. aeruginosa PA14 were examined. Three Carex plant extracts at a concentration of 200 µg/mL inhibited P. aeruginosa biofilm formation by >80% without affecting planktonic cell growth. In the most active extract of Carex pumila , resveratrol dimer ε-viniferin was one of the main antibiofilm compounds against P. aeruginosa. Interestingly, ε-viniferin at 10 µg/mL inhibited biofilm formation of enterohemorrhagic Escherichia coli O157:H7 by 98%. Although Carex extracts and trans-resveratrol are known to possess antimicrobial activity, this study is the first to report that C. pumila extract and ε-viniferin have antibiofilm activity against P. aeruginosa and E. coli O157:H7.

Anti-biofilm activities of quercetin and tannic acid against Staphylococcus aureus.

Staphylococcus aureus is a leading cause of nosocomial infections because of its resistance to diverse antibiotics. The formation of a biofilm is one of the mechanisms of drug resistance in S. aureus. The anti-biofilm abilities of 498 plant extracts against S. aureus were examined. Seventy-two plant extracts belonging to 59 genera and 38 families were found to significantly inhibit the formation of biofilms of S. aureus without affecting the growth of planktonic cells. The most active extract, from Alnus japonica, inhibited the formation of biofilms by three S. aureus strains by >70% at 20 µg ml(-1). Transcriptional analyses showed that extract of A. japonica repressed the intercellular adhesion genes icaA and icaD most markedly. Quercetin and tannic acid are major anti-biofilm compounds in the extract of A. japonica. Additionally, the extract of A. japonica and its component compound quercetin, reduced hemolysis by S. aureus. This phenomenon was not observed in the treatment with tannic acid. This study suggests that various plant extracts, such as quercetin and tannic acid, could be used to inhibit the formation of recalcitrant biofilms of S. aureus.

Indole and 7-benzyloxyindole attenuate the virulence of Staphylococcus aureus.

Human pathogens can readily develop drug resistance due to the long-term use of antibiotics that mostly inhibit bacterial growth. Unlike antibiotics, antivirulence compounds diminish bacterial virulence without affecting cell viability and thus, may not lead to drug resistance. Staphylococcus aureus is a major agent of nosocomial infections and produces diverse virulence factors, such as the yellow carotenoid staphyloxanthin, which promotes resistance to reactive oxygen species (ROS) and the host immune system. To identify novel antivirulence compounds, bacterial signal indole present in animal gut and diverse indole derivatives were investigated with respect to reducing staphyloxanthin production and the hemolytic activity of S. aureus. Treatment with indole or its derivative 7-benzyloxyindole (7BOI) caused S. aureus to become colorless and inhibited its hemolytic ability without affecting bacterial growth. As a result, S. aureus was more easily killed by hydrogen peroxide (H2O2) and by human whole blood in the presence of indole or 7BOI. In addition, 7BOI attenuated S. aureus virulence in an in vivo model of nematode Caenorhabditis elegans, which is readily infected and killed by S. aureus. Transcriptional analyses showed that both indole and 7BOI repressed the expressions of several virulence genes such as α-hemolysin gene hla, enterotoxin seb, and the protease genes splA and sspA and modulated the expressions of the important regulatory genes agrA and sarA. These findings show that indole derivatives are potential candidates for use in antivirulence strategies against persistent S. aureus infection.