Pleiotropic Drug Resistance Transporter Research Articles

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two Orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, Oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three Pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

Genome-Wide Identification of Pleiotropic Drug Resistance (PDR) Transporters in Salix purpurea and Expression Analysis in Response to Various Heavy Metal Stresses

Pleiotropic drug resistance (PDR) transporters, which are part of the ABCG subfamily of ATP-binding cassette (ABC) transporters, have been found to be involved in heavy metal tolerance. Salix species (willow) is widely regarded as a perfect candidate for phytoremediation of heavy metals because of its substantial biomass, strong tolerance, and remarkable capacity to accumulate heavy metals. However, the phylogeny and mechanisms underlying the response to heavy metals within the PDR family in willow have yet to be determined. In this study, we discovered and valuated a total of 21 PDR genes in the genome of Salix purpurea. The phylogenetic relationships of these genes were used to classify them into five major clades. The SpPDRs exhibited variations in exon-intron distribution patterns and gene lengths across different branches. Cis-acting elements linked to stress response, drought induction, low temperature, and defense response were discovered in the promoters of PDRs. Significant variations in the transcription levels of various PDR genes were observed across different tissues under heavy metal stress, with distinct heavy metals regulating different PDR members. In roots, PDR4 and PDR21 exhibited high expression levels. Meanwhile, PDR7 and PDR17 showed similar transcription patterns across all analyzed tissues. Furthermore, there was a significant and positive correlation between PDR5 and PDR16, whereas a significant and negative correlation was detected between PDR3 and PDR9, suggesting that the response of PDR members to heavy metals is complex and multifaceted. These findings will establish a vital basis for comprehending the biological functionalities of PDR genes, specifically their involvement in the regulation of willow’s tolerance to heavy metals.

Tyrosol induces multiple drug resistance in yeast Saccharomyces cerevisiae.

In yeast, multiple (pleiotropic) drug resistance (MDR) transporters efflux xenobiotics from the cytoplasm to the environment. Additionally, upon the accumulation of xenobiotics in the cells, MDR genes are induced. At the same time, fungal cells can produce secondary metabolites with physico-chemical properties similar to MDR transporter substrates. Nitrogen limitation in yeast Saccharomyces cerevisiae leads to the accumulation of phenylethanol, tryptophol, and tyrosol, which are products of aromatic amino acid catabolism. In this study, we investigated whether these compounds could induce or inhibit MDR in yeast. Double deletion of PDR1 and PDR3 genes, which are transcription factors that upregulate the expression of PDR genes, reduced yeast resistance to high concentrations of tyrosol (4-6 g/L) but not to the other two tested aromatic alcohols. PDR5 gene, but not other tested MDR transporter genes (SNQ2, YOR1, PDR10, PDR15) contributed to yeast resistance to tyrosol. Tyrosol inhibited the efflux of rhodamine 6G (R6G), a substrate for MDR transporters. However, preincubating yeast cells with tyrosol induced MDR, as evidenced by increased Pdr5-GFP levels and reduced yeast ability to accumulate Nile red, another fluorescent MDR-transporter substrate. Moreover, tyrosol inhibited the cytostatic effect of clotrimazole, the azole antifungal. Our results demonstrate that a natural secondary metabolite can modulate yeast MDR. We speculate that intermediates of aromatic amino acid metabolites coordinate cell metabolism and defense mechanisms against xenobiotics.

A Pleiotropic Drug Resistance Transporter TaABCG36 Contributes to Defense against Puccinia triticina in Triticum aestivum

ABC transporters play important roles in plant growth and resistance to abiotic and biotic stresses. Here, we showed that the TaABCG36 gene positively regulates leaf rust resistance in the wheat line Thatcher + Lr19 (TcLr19) when challenged with an avirulent pathotype of Puccinia triticina (Pt). The TaABCG36 gene was cloned from genomic DNA and cDNA from wheat line TcLr19. The clone was 6730 bp in gDNA and 4365 bp in cDNA for this gene. It encoded an ABC transporter with 1454 amino acids in length. BLASTp analysis indicated a considerable identity ABC transporter G family member 36 with Aegilops tauschii subsp. strangulata, Triticum dicoccoides, and T. aestivum; thus, we named the gene TaABCG36. TaABCG36 was proved to be a plasma transmembrane protein by bioinformatic analysis and subcellular localization of the TaABCG36–GFP fusion protein. The expression of TaABCG36 in wheat leaves reached a peak at 72 h post-inoculation by Pt avirulence pathotype, and the expression was also induced by phytohormone treatments of salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA). Three fragments (V1–V3) of the TaABCG36 gene were introduced to the BSMV-VIGS vector and, thus, silenced the expression of TaABCG36 in the wheat line TcLr19. All the three BSMV:VIGS-infected plants showed reaction type “3” to Pt pathotype THTS, which was fully avirulent on TcLr19 (infection type “0”). Histopathological observation showed that silencing of TaABCG36 facilitated the formation of haustorial mother cells (HMC) and mycelial growth, implying that TaABCG36 plays a positive role in the response of TcLr19 against THTS. These results provide molecular insight into the interaction between Pt and its wheat host and identify a potential target for engineering resistance in wheat to damaging pathogen of Pt.

The Role of Plasma Membrane Pleiotropic Drug Resistance Transporters in the Killer Activity of Debaryomyces hansenii and Wickerhamomyces anomalus Toxins.

The killer strains of Debaryomyces hansenii and Wickerhamomyces anomalus species secrete antimicrobial proteins called killer toxins which are active against selected fungal phytopathogens. In our research, we attempted to investigate the role of plasma membrane pleiotropic drug resistance (PDR) transporters (Pdr5p and Snq2p) in the mechanism of defense against killer toxins. Saccharomyces cerevisiae mutant strains with strengthened or weakened pleiotropic drug resistance due to increased or reduced number of mentioned PDR efflux pumps were tested for killer toxin susceptibility. The present study demonstrates the influence of the Snq2p efflux pump in immunity to W. anomalus BS91 killer toxin. It was also shown that the activity of killer toxins of D. hansenii AII4b, KI2a, MI1a and CBS767 strains is regulated by other transporters than those influencing W. anomalus killer toxin activity. In turn, this might be related to the functioning of the Pdr5p transporter and a complex cross-talk between several regulatory multidrug resistance networks. To the best of our knowledge, this is the first study that reports the involvement of PDR transporters in the cell membrane of susceptible microorganisms in resistance to killer yeasts’ toxins.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker’s yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum.

Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum. An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1, overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1. Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species.

Multidrug Resistance in Mammals and Fungi-From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms.

Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure–function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

Characterization of Three Pleiotropic Drug Resistance Transporter Genes and Their Participation in the Azole Resistance of Mucor circinelloides.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

Ferulic acid, an abundant maize phenolic, regulates ABC and MFS transporter gene expression in the maize pathogen Cochliobolus heterostrophus

Plant pathogens encounter pre-existing and induced defense compounds when they invade the host. Pathogen ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters can provide protection during infection through efflux removal of plant defense compounds from the cell. In the current work, we identified a total of 973 transporter genes, including 232 MFS and 39 ABC transporters, in the genome of the southern corn leaf blight pathogen Cochliobolus heterostrophus. We compared these genes with a recently published transcriptomic data set from C. heterostrophus exposed to the phenolic maize defense compound ferulic acid (FA) and identified four transporter gene families that were significantly (P < 0.05) enriched among genes upregulated by FA, including MFS and ABC transporters, the mitochondrial carrier family and the fatty acid transporter family. Phylogenetic analyses further classified most FA-upregulated ABC transporters into subgroups G (pleiotropic drug resistance transporters), B (multi-drug resistance transporters) and C (multi-drug resistance-associated transporters). Out of the 74 MFS transporter genes induced by FA, 29 were classified as members of the 2.A.1.2 Drug:H + Antiporter-1 (DHA1) family. The results support the idea that efflux of plant defense compounds by drug resistance membrane transporters contributes to pathogen virulence. The provided MFS and ABC transporter classification should be readily extendable to other Dothideomycete pathogen genomes.

Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1.

ABSTRACTPleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae. The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX2[3]CPX3NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an “almost cysteine-less,” yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

Comparative genomics highlights the importance of drug efflux transporters during evolution of mycoparasitism in Clonostachys subgenus Bionectria (Fungi, Ascomycota, Hypocreales).

Various strains of the mycoparasitic fungal species Clonostachys rosea are used commercially as biological control agents for the control of fungal plant diseases in agricultural crop production. Further improvements of the use and efficacy of C. rosea in biocontrol require a mechanistic understanding of the factors that determines the outcome of the interaction between C. rosea and plant pathogenic fungi. Here, we determined the genome sequences of 11 Clonostachys strains, representing five species in Clonostachys subgenus Bionectria, and performed a comparative genomic analysis with the aim to identify gene families evolving under selection for gene gains or losses. Several gene families predicted to encode proteins involved in biosynthesis of secondary metabolites, including polyketide synthases, nonribosomal peptide syntethases and cytochrome P450s, evolved under selection for gene gains (p ≤ .05) in the Bionectria subgenus lineage. This was accompanied with gene copy number increases (p ≤ .05) in ATP‐binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters predicted to contribute to drug efflux. Most Clonostachys species were also characterized by high numbers of auxiliary activity (AA) family 9 lytic polysaccharide monooxygenases, AA3 glucose–methanol–choline oxidoreductases and additional carbohydrate‐active enzyme gene families with putative activity (or binding) towards xylan and rhamnose/pectin substrates. Particular features of the C. rosea genome included expansions (p ≤ .05) of the ABC‐B4 multidrug resistance transporters, the ABC‐C5 multidrug resistance‐related transporters and the 2.A.1.3 drug:H + antiporter‐2 MFS drug resistance transporters. The ABC‐G1 pleiotropic drug resistance transporter gene abcG6 in C. rosea was induced (p ≤ .009) by exposure to the antifungal Fusarium mycotoxin zearalenone (1121‐fold) and various fungicides. Deletion of abcG6 resulted in mutants with reduced (p < .001) growth rates on media containing the fungicides boscalid, fenhexamid and iprodione. Our results emphasize the role of biosynthesis of, and protection against, secondary metabolites in Clonostachys subgenus Bionectria.

AaABCG40 Enhances Artemisinin Content and Modulates Drought Tolerance in Artemisia annua.

The phytohormone Abscisic acid (ABA) regulates plant growth, development, and responses to abiotic stresses, including senescence, seed germination, cold stress and drought. Several kinds of researches indicate that exogenous ABA can enhance artemisinin content in A. annua. Some transcription factors related to ABA signaling are identified to increase artemisinin accumulation through activating the artemisinin synthase genes. However, no prior study on ABA transporter has been performed in A. annua. Here, we identified a pleiotropic drug resistance (PDR) transporter gene AaPDR4/AaABCG40 from A. annua. AaABCG40 was expressed mainly in roots, leaves, buds, and trichomes. GUS activity is primarily observed in roots and the vascular tissues of young leaves in proAaABCG40: GUS transgenic A. annua plants. When AaABCG40 was transferred into yeast AD12345678, yeasts expressing AaABCG40 accumulated more ABA than the control. The AaABCG40 overexpressing plants showed higher artemisinin content and stronger drought tolerance. Besides, the expression of CYP71AV1 in OE-AaABCG40 plants showed more sensitivity to exogenous ABA than that in both wild-type and iAaABCG40 plants. According to these results, they strongly suggest that AaABCG40 is involved in ABA transport in A. annua.

Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata

Microbial cells sense the presence of xenobiotics and, in response, upregulate genes involved in pleiotropic drug resistance (PDR). In yeast, PDR activation to a major extent relies on the transcription factor Pdr1. In addition, many xenobiotics induce oxidative stress, which may upregulate PDR independently of Pdr1 activity. Mitochondria are important sources of reactive oxygen species under stressful conditions. To evaluate the relevance of this redox pathway, we studied the activation of PDR in the yeast Candida glabrata, which we treated with a mitochondrially targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium and dodecyltriphenylphosphonium (C12TPP) as a control. We found that both compounds induced activation of PDR genes and decreased the intracellular concentration of the PDR transporter substrate Nile red. Interestingly, the deletion of PDR transporter gene CDR1 inhibited the decrease in Nile red accumulation induced by antioxidant plastoquinonyl-decyl-triphenylphosphonium but not that by C12TPP. Moreover, antioxidant alpha-tocopherol inhibited C12TPP-mediated activation of PDR in Δcdr1 but not in the wild-type strain. Furthermore, pre-incubation of yeast cells with low concentrations of hydrogen peroxide induced a decrease in the intracellular concentration of Nile red in Δcdr1 and Δpdr1 as well as in control cells. Deletion of PDR1 inhibited the C12TPP-induced activation of CDR1 but not that of FLR1, which is a redox-regulated PDR transporter gene. It appears that disruption of the PDR1/CDR1 regulatory circuit makes auxiliary PDR regulation mechanisms crucial. Our data suggest that redox regulation of PDR is dispensable in wild-type cells because of redundancy in the activation pathways, but is manifested upon deletion of CDR1.

PDR-like ABC systems in pathogenic fungi

ABC transporters of the Pleiotropic Drug Resistance (PDR) family are the main actors of antifungal resistance in pathogenic fungi. While their involvement in clinical resistant strains has been proven, their transport mechanism remains unclear. Notably, one hallmark of PDR transporters is their asymmetry, with one canonical nucleotide-binding site capable of ATP hydrolysis while the other site is not. Recent publications reviewed here show that the so-called “deviant” site is of crucial importance for drug transport and is a step towards alleviating the mystery around the existence of non-catalytic binding sites.

The Arabidopsis Pleiotropic Drug Resistance Transporters PEN3 and PDR12 Mediate Camalexin Secretion for Resistance to Botrytis cinerea.

Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

Cloning and expression of selected ABC transporters from the Arabidopsis thaliana ABCG family in Pichia pastoris.

Phytohormones play a major role in plant growth and development. They are in most cases not synthesized in their target location and hence need to be transported to the site of action, by for instance ATP-binding cassette transporters. Within the ATP-binding cassette transporter family, Pleiotropic Drug Resistance transporters are known to be involved in phytohormone transport. Interestingly, PDRs are only present in plants and fungi. In contrast to fungi, there are few biochemical studies of plant PDRs and one major reason is that suitable overexpression systems have not been identified. In this study, we evaluate the expression system Pichia pastoris for heterologous overexpression of PDR genes of the model plant Arabidopsis thaliana. We successfully cloned and expressed the potential phytohormone transporters PDR2 and PDR8 in P. pastoris. Sucrose gradient centrifugation confirmed that the overexpressed proteins were correctly targeted to the plasma membrane of P. pastoris and initial functional studies demonstrated ATPase activity for WBC1. However, difficulties in cloning and heterologous overexpression might be particular obstacles of the PDR family, since cloning and overexpression of White Brown Complex 1, a half-size transporter of the same ABCG subfamily with comparable domain organization, was more easily achieved. We present strategies and highlight critical factors to successfully clone plant PDR genes and heterologously expressed in P. pastoris.

Information theoretic measures and mutagenesis identify a novel linchpin residue involved in substrate selection within the nucleotide-binding domain of an ABCG family exporter Cdr1p

ABC transporters are membrane-bound pumps composed of two major domains: the transmembrane domain(s) (TMDs) and the nucleotide-binding domain(s) (NBDs). Sequence analyses of the NBDs of key ABC exporters revealed a residue position within the H-loop to be differentially conserved in the ABCG family, wherein there lies glutamine instead of positively charged arginine/lysine as in non-ABCG members. Consequently, contrasting NBD sequences of fungal Pleiotropic Drug Resistance transporters (PDR/ABCG) with that of Cholesterol/Phospholipid and Retinal (CPR/ABCA) Flippase family revealed a high Cumulative Relative Entropy (CRE) score of this residue position implying its family-specific functional significance. Further, substitution of the glutamine by arginine in both the NBDs of a representative PDR/ABCG member, (Candida drug resistance 1 protein) Cdr1p led to selective susceptibility of the Saccharomyces cerevisiae strains overexpressing the corresponding mutant proteins (Q362R and Q1060R) towards antifungal substrates without any impact on the ATPase activity. Consistent with the findings from previous studies on H-loop motif of fungal PDR transporters, the current report points towards a role of the glutamine residue within both canonical and divergent H-loop of Cdr1p in conferring substrate selection in a precisely identical manner.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.