Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.

Rapid necrosis II: physiological and molecular analysis of 2,4-D resistance in Sumatran fleabane (Conyza sumatrensis)

In 2015, plants of Sumatran fleabane [Conyza sumatrensis (Retz.) E. Walker] were identified in a crop field with an unusual rapid necrosis herbicide symptom after application of 2,4-D. An initial study identified that the symptoms began about 2 h after herbicide application, the resistant factor is high (resistance factor = 19), and the resistance decreased at low light. The mechanism of resistance is not known yet, but the symptomatology suggests it may be related to reduced translocation, ATP-binding cassette class B (ABCB) transporters, changes on auxin perception genes or induction of genes involved in response to pathogens and abiotic stresses. The objective of this study was to investigate the mechanisms involved in the resistance to 2,4-D caused by rapid necrosis using inhibitors of enzymes involved in detoxification and carriers. Neither the inhibitors of ABCB and auxin transporters TIBA, NPA, verapamil and orthovanadate, nor the inhibitors of detoxifying enzymes, as malathion, NBD-Cl and imidazole, avoided the rapid necrosis phenotype. However, orthovanadate and sodium azide (possibly related with auxin transport) were able to partially reduce oxidative stress in leaf disc. The expression of ABCM10 (an ABCD transporter gene), TIR1_1 (an auxin receptor gene) and CAT4 (an amino acid transporter gene) was quickly reduced after 2,4-D application in the resistant accession. Contrary to our hypothesis, LESION SIMULATING DISEASE RESISTANCE 1_3 (LSD1_3) expression increased in response to 2,4-D. LSD1_3 is important for the response to pathogen and abiotic stresses. The rapid necrosis mechanism is not related to 2,4-D detoxification but might be related to changes in the TIR receptor or auxin transport. Mutations in other transporters or in proteins involved in abiotic and pathogen stresses cannot be ruled out.

Diketopiperazine Modulates Arabidopsis thaliana Root System Architecture by Promoting Interactions of Auxin Receptor TIR1 and IAA7/17 Proteins

ABSTRACT Plants can detect the quorum sensing (QS) signaling molecules of microorganisms, such as amino acids, fat derivatives and diketopiperazines (DKPs), thus allowing the exchange information to promote plant growth and development. Here, we evaluated the effects of 12 synthesized DKPs on Arabidopsis thaliana roots and studied their underlying mechanisms of action. Results showed that, as QS signal molecules, the DKPs promoted lateral root development and root hair formation in A.thaliana to differing degrees. The DKPs enhanced the polar transport of the plant hormone auxin from the shoot to root and triggered the auxin-responsive protein IAA7/17 to decrease the auxin response factor, leading to the accumulation of auxin at the root tip and accelerated root growth. In addition, the DKPs induced the development of lateral roots and root hair in the A. thaliana root system architecture via interference with auxin receptor transporter inhibitor response protein 1 (TIR1). A series of TIR1 sites that potentially interact with DKPs were also predicted using molecular docking analysis. Mutations of these sites inhibited the phosphorylation of TIR1 after DKP treatment, thereby inhibiting lateral root formation, especially TIR1-1 site. This study identified several DKP signal molecules in the QS system that can promote the expression of auxin response factors ARF7/19 via interactions of TIR1 and IAA7/17 proteins, thus promoting plant growth and development.

The Lotus japonicus AFB6 Gene Is Involved in the Auxin Dependent Root Developmental Program

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N2-fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6. A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.

In silico promoter and expression analyses of rice Auxin Binding Protein 57 (ABP57)

Auxin Binding Protein 57 (ABP57) is one of the molecular components involved in rice response to abiotic stress. The ABP57 gene encodes an auxin receptor which functions in activating the plasma membrane H+-ATPase. Biochemical properties of ABP57 have been characterized; however, the function of ABP57, particularly on stress and hormone responses is still limited. This study was conducted to understand the regulation of ABP57 expression under abiotic stress. Thus, in silico identification of cis-acting regulatory elements (CAREs) in the promoter region of ABP57 was performed. Several motifs and transcription factor binding site (TFBS) that are involved in abiotic stress such as ABRE, DRE, AP2/EREBP, WRKY and NAC were identified. Next, expression analysis of ABP57 under drought, salt, auxin (IAA) and abscisic acid (ABA) was conducted by reverse transcription-PCR (RT-PCR) to verify the effect of these treatments on ABP57 transcript level. ABP57 was expressed at different levels in the shoot and root under drought conditions, and its expression was increased under IAA and ABA treatments. Moreover, our results showed that ABP57 expression in the root was more responsive to drought, auxin and ABA treatments compared to its transcript in the shoot. This finding suggests that ABP57 is a drought-responsive gene and possibly regulated by IAA and ABA.

Variation of Glucosinolate Contents in Clubroot-Resistant and -Susceptible Brassica napus Cultivars in Response to Virulence of Plasmodiophora brassicae

The present study investigated the changes in total and individual glucosinolates (GSLs) in roots and leaves of different clubroot-resistant and -susceptible oilseed rape cultivars following artificial inoculation with Plasmodiophora brassicae isolates with different virulence. The results showed significant differences in clubroot incidence and severity as well as in the amount of total and individual glucosinolates between oilseed rape cultivars in response to virulence of the pathogen. Single among with total aliphatic and total indolic glucosinolate contents were significantly lower in leaves of susceptible cultivars compared to resistant ones due to the infection. Similarly, single and total aliphatic as well as indolic glucosinolate contents in roots were lower in susceptible cultivars compared to resistant cultivars analyzed. The different isolates of P. brassicae seem to differ in their ability to reduce gluconasturtiin contents in the host. The more aggressive isolate P1 (+) might be able to suppress gluconasturtiin synthesis of the host in a more pronounced manner compared to the isolate P1. A possible interaction of breakdown products of glucobrassicin with the auxin receptor transport inhibitor response 1 (TIR1) is hypothesized and its possible effects on auxin signaling in roots and leaves of resistant and susceptible cultivars is discussed. A potential interplay between aliphatic and indolic glucosinolates that might be involved in water homeostasis in resistant cultivars is explained.

The volatile cedrene from plant beneficial Trichoderma guizhouense modulates Arabidopsis root development through auxin transport and signaling

Rhizosphere microorganisms interact with plant roots by producing chemical signals to regulate root development. However, the involved distinct bioactive compounds and the signal transduction pathways are remaining to be identified. Here, we show that sesquiterpenes (SQTs) are the main volatile compounds produced by plant beneficial Trichoderma guizhouense NJAU 4742, inhibition of SQTs synthesis in this strain indicated their involvement in plant-fungus cross-kingdom signaling. SQTs component analysis further identified the cedrene, a high abundant SQT in strain NJAU 4742, could stimulate plant growth and root development. Genetic analysis and auxin transport inhibition showed that auxin receptor TIR1, AFB2, auxin-responsive protein IAA14, and transcription factor ARF7, ARF19 affect the response of lateral roots to cedrene. Moreover, auxin influx carrier AUX1, efflux carrier PIN2 were also indispensable for cedrene-induced lateral root formation. Confocal imaging showed that cedrene affected the expression of pPIN2:PIN2:GFP and pPIN3:PIN3:GFP, which may be related to the effect of cedrene on root morphology. These results suggest that a novel SQT molecule from plant beneficial T. guizhouense can regulate plant root development through auxin transport and signaling.

Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

ABSTRACTAuxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics or transcription, that are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complexes subunits in mouse embryonic stem cells (mRad21, mSMC2, mCapH, mCapH2) and human haploid HAP1 line (hRad21, hSMC2). Double antibiotic selection helped to achieve high homozygous AID targeting efficiency for all genes, ranging from 11 to 77%. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to mosaic expression of AFBs, which also tends to decline with passages in the absence of constant puromycin selection, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that OsTIR1 system showed weak dynamics of protein degradation. At the same time, AtAFB2 approach was very efficient even in random integration. Other factors such as degradation dynamics and low basal depletion were also in favor of AtAFB2 system. Our main conclusion is that repeated addition of puromycin to culture medium prevents AtAFB2 silencing and restores auxin sensitivity, facilitating robust protein degradation. We hope that our report will be useful for researchers that plan to establish AID method in their lab.

Bioprospecting Fluorescent Plant Growth Regulators from Arabidopsis to Vegetable Crops

The phytohormone auxin is involved in almost every process of a plant’s life, from germination to plant development. Nowadays, auxin research connects synthetic chemistry, plant biology and computational chemistry in order to develop innovative and safe compounds to be used in sustainable agricultural practice. In this framework, we developed new fluorescent compounds, ethanolammonium p-aminobenzoate (HEA-pABA) and p-nitrobenzoate (HEA-pNBA), and investigated their auxin-like behavior on two main commercial vegetables cultivated in Europe, cucumber (Cucumis sativus) and tomato (Solanumlycopersicum), in comparison to the model plant Arabidopsis (Arabidopsis thaliana). Moreover, the binding modes and affinities of two organic salts in relation to the natural auxin indole-3-acetic acid (IAA) into TIR1 auxin receptor were investigated by computational approaches (homology modeling and molecular docking). Both experimental and theoretical results highlight HEA-pABA as a fluorescent compound with auxin-like activity both in Arabidopsis and the commercial cucumber and tomato. Therefore, alkanolammonium benzoates have a great potential as promising sustainable plant growth stimulators to be efficiently used in vegetable crops.