Physiological implications of SWEETs in plants and their potential applications in improving source–sink relationships for enhanced yield

The sugars will eventually be exported transporters (SWEET) family of transporters in plants is identified as a novel class of sugar carriers capable of transporting sugars, sugar alcohols and hormones. Functioning in intercellular sugar transport, SWEETs influence a wide range of physiologically important processes. SWEETs regulate the development of sink organs by providing nutritional support from source leaves, responses to abiotic stresses by maintaining intracellular sugar concentrations, and host–pathogen interactions through the modulation of apoplastic sugar levels. Many bacterial and fungal pathogens activate the expression of SWEET genes in species such as rice and Arabidopsis to gain access to the nutrients that support virulence. The genetic manipulation of SWEETs has led to the generation of bacterial blight (BB)-resistant rice varieties. Similarly, while the overexpression of the SWEETs involved in sucrose export from leaves and pathogenesis led to growth retardation and yield penalties, plants overexpressing SWEETs show improved disease resistance. Such findings demonstrate the complex functions of SWEETs in growth and stress tolerance. Here, we review the importance of SWEETs in plant–pathogen and source–sink interactions and abiotic stress resistance. We highlight the possible applications of SWEETs in crop improvement programmes aimed at improving sink and source strengths important for enhancing the sustainability of yield. We discuss how the adverse effects of the overexpression of SWEETs on plant growth may be overcome.     

Cellular export of sugars and amino acids: role in feeding other cells and organisms

Sucrose, hexoses, and raffinose play key roles in the plant metabolism. Sucrose and raffinose, produced by photosynthesis, are translocated from leaves to flowers, developing seeds and roots. Translocation occurs in the sieve elements or sieve tubes of angiosperms. But how is sucrose loaded into and unloaded from the sieve elements? There seem to be two principal routes: one through plasmodesmata and one via the apoplasm. The best-studied transporters are the H⁺/SUCROSE TRANSPORTERs (SUTs) in the sieve element-companion cell complex. Sucrose is delivered to SUTs by SWEET sugar uniporters that release these key metabolites into the apoplasmic space. The H⁺/amino acid permeases and the UmamiT amino acid transporters are hypothesized to play analogous roles as the SUT-SWEET pair to transport amino acids. SWEETs and UmamiTs also act in many other important processes—for example, seed filling, nectar secretion, and pollen nutrition. We present information on cell type-specific enrichment of SWEET and UmamiT family members and propose several members to play redundant roles in the efflux of sucrose and amino acids across different cell types in the leaf. Pathogens hijack SWEETs and thus represent a major susceptibility of the plant. Here, we provide an update on the status of research on intercellular and long-distance translocation of key metabolites such as sucrose and amino acids, communication of the plants with the root microbiota via root exudates, discuss the existence of transporters for other important metabolites and provide potential perspectives that may direct future research activities.

An update on intercellular and long-distance translocation of sugars and amino acids, including plant-root microbiota communication, other metabolite transporters is provided, and perspectives are discussed.     

SWEET蛋白家族研究进展

SWEET是新发现的一类具有7次跨膜?-螺旋的糖运输蛋白,它们由2个重复的具有3次跨膜?-螺旋的MtN3 motif和一个起连接作用的跨膜?-螺旋组成.SWEET广泛存在于真核单细胞生物、高等植物以及动物中.它们在生殖发育、植物与微生物的相互作用、植物的逆境反应及衰老等许多方面起重要作用.最近的研究显示,原核生物中存在与真核生物SWEET类似的、只含有一个3次跨膜?-螺旋的蛋白,这些蛋白属于MtN3或PQ-Loop家族.从慢生根瘤菌中克隆的SWEET同源蛋白BjSemiSWEET1和已经鉴定的部分真核生物SWEET蛋白一样具有运输蔗糖的能力,这个结果与其他相关研究一起暗示真核生物7次跨膜?-螺旋的糖或氨基酸运输蛋白可能由原核生物中3次跨膜?-螺旋的小分子蛋白通过复制或横向基因转移融合进化而来,并且它们在行使功能时可能形成和其他许多膜转运蛋白相似的、具有12次跨膜结构的功能单位.对SWEET的研究将为揭示多种生命现象提供重要线索.     

木薯SWEETs基因家族生物信息学及表达特性研究

SWEET (sugars will eventually be exported transporters)是植物中新发现的一类编码糖转运蛋白的基因,它在植物生长发育及糖代谢过程中发挥重要作用。该基因家族在木薯(Manihot esculenta)中尚未有详细的报道。本研究从Phytozome数据库获得了28个木薯SWEET候选基因并对其进行生物信息学分析,在华南124的木薯苗中通过荧光定量实验检测SWEET基因在旱胁迫下的表达水平。结果发现木薯SWEET基因被分为4簇,主要分布在第6条和第14条染色体上,编码234 aa与302 aa之间的氨基酸序列;木薯SWEET基因家族的表达在旱胁迫条件下发生了变化,其中明显上调的基因有9个,包括MeSWEET1b、MeSWEET2a、MeSWEET6、MeSWEET9a、MeSWEET9b、MeSWEET12、MeSWEET15a、MeSWEET15b和MeSWEET16c;而表达量明显下调的基因也有9个,为MeSWEET2b、MeSWEET3b、MeSWEET4、MeSWEET7、MeSWEET11、MeSWEET16a、MeSWEET16b、MeSWEET17a和MeSWEET17c。这些结果为进一步阐明SWEET基因家族在木薯中的功能提供理论依据。     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

dbSWEET: An Integrated Resource for SWEET Superfamily to Understand,Analyze and Predict the Function of Sugar Transporters in Prokaryotes and Eukaryotes

SWEET (Sweet Will Eventually be Exported Transporter) proteins have been recently discovered and form one of the three major families of sugar transporters. Homologs of SWEET are found in both prokaryotes and eukaryotes. Bacterial SWEET homologs have three transmembrane segments forming a triple-helical bundle and the functional form is dimers. Eukaryotic SWEETs have seven transmembrane helical segments forming two triple-helical bundles with a linker helix. Members of SWEET homologs have been shown to be involved in several important physiological processes in plants. However, not much is known regarding the biological significance of SWEET homologs in prokaryotes and in mammals. We have collected more than 2000 SWEET homologs from both prokaryotes and eukaryotes. For each homolog, we have modeled three different conformational states representing outward open, inward open and occluded states. We have provided details regarding substrate-interacting residues and residues forming the selectivity filter for each SWEET homolog. Several search and analysis options are available. The users can generate a phylogenetic tree and structure-based sequence alignment for selected set of sequences. With no metazoan SWEETs functionally characterized, the features observed in the selectivity filter residues can be used to predict the potential substrates that are likely to be transported across the metazoan SWEETs. We believe that this database will help the researchers to design mutational experiments and simulation studies that will aid to advance our understanding of the physiological role of SWEET homologs. This database is freely available to the scientific community at http://bioinfo.iitk.ac.in/bioinfo/dbSWEET/Home.     

A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.     

Plant integrity: An important factor in plant-pathogen interactions

The effect of plant integrity and of aboveground-belowground defense signaling on plant resistance against pathogens and herbivores is emerging as a subject of scientific research. There is increasing evidence that plant defense responses to pathogen infection differ between whole intact plants and detached leaves. Studies have revealed the importance of aboveground-belowground defense signaling for plant defenses against herbivores, while our studies have uncovered that the roots as well as the plant integrity are important for the resistance of the potato cultivar Sarpo Mira against the hemibiotrophic oomycete pathogen Phytophthora infestans. Furthermore, in the Sarpo Mira–P. infestans interactions, the plant’s meristems, the stalks or both, seem to be associated with the development of the hypersensitive response and both the plant’s roots and shoots contain antimicrobial compounds when the aerial parts of the plants are infected. Here, we present a short overview of the evidence indicating the importance of plant integrity on plant defense responses.     

Female Salix viminalis are more severely infected by Melampsora spp. but neither sex experiences associational effects

Associational effects of plant genotype or species on plant biotic interactions are common, not least for disease spread, but associational effects of plant sex on interactions have largely been ignored. Sex in dioecious plants can affect biotic interactions with herbivores and pollinators; however, its effects on plant–pathogen interactions are understudied and associational effects are unknown. In a replicated field experiment, we assessed Melampsora spp. leaf rust infection in monosexual and mixed sex plots of dioecious Salix viminalis L. to determine whether plant sex has either direct or associational effects on infection severity. We found no differences in Melampsora spp. infection severity among sexual monocultures and mixtures in our field experiment. However, female plants were overall more severely infected. In addition, we surveyed previous studies of infection in S. viminalis clones and reevaluated the studies after we assigned sex to the clones. We found that females were generally more severely infected, as in our field study. Similarly, in a survey of studies on sex‐biased infection in dioecious plants, we found more female‐biased infections in plant–pathogen pairs. We conclude that there was no evidence for associational plant sex effects of neighboring conspecifics for either females or males on infection severity. Instead, plant sex effects on infection act at an individual plant level. Our findings also suggest that female plants may in general be more severely affected by fungal pathogens than males.     

SWEET17, a Facilitative Transporter,Mediates Fructose Transport across the Tonoplast of Arabidopsis Roots and Leaves

Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-β-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [¹⁴C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH₄Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.Sugars are main energy sources for generating ATP, major precursors to various storage carbohydrates as well as key signaling molecules important for normal growth in higher plants (Rolland et al., 2006). Depending on the metabolic demand, sugars are translocated over long distances or stored locally. SWEET (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS) and SUC/SUT (for Sucrose transporter/Sugar transporter)-type transporters are responsible for transfer of Suc from the phloem parenchyma into the sieve element companion cell complex for long-distance translocation (Riesmeier et al., 1992; Sauer, 2007; Kühn and Grof, 2010; Chen et al., 2012). Suc or hexoses derived from Suc hydrolysis in the cell wall are then taken up into sink cells by SUT (Braun and Slewinski, 2009) or monosaccharide transporters, such as sugar transporter1 (Sauer et al., 1990; Pego and Smeekens, 2000; Sherson et al., 2003). Alternatively, sugars are thought to move between cells via plasmodesmata (Voitsekhovskaja et al., 2006; Ayre, 2011). Major sugar storage pools within plant cells are soluble sugars stored in the vacuole, starch in plastids, and lipids in oil bodies.Vacuoles, which can account for approximately 90% of the cell volume (Winter et al., 1993), play central roles in temporary and long-term storage of soluble sugars (Martinoia et al., 2007; Etxeberria et al., 2012). Some agriculturally important crops such as sugar beet (Beta vulgaris; Leigh, 1984; Getz and Klein, 1995), citrus (Citrus spp.; Echeverria and Valich, 1988), sugarcane (Saccharum officinarum; Thom et al., 1982), and carrot (Daucus carota; Keller, 1988) can store considerable amounts (>10% of plant dry weight) of Suc, Glc, or Fru in vacuoles of the storage parenchyma. Due to a high capacity of vacuoles for storing sugars, vacuolar sugars can serve as an important carbohydrate source during energy starvation, e.g. after starch has been exhausted (Echeverria and Valich, 1988), as well as for the production of other compounds (e.g. osmoprotectants). Sugars are known to regulate photosynthesis; therefore, the release of sugars from vacuoles could be important for modulating photosynthesis (Kaiser and Heber, 1984). Moreover, vacuole-derived sugars are commercially used to produce biofuels, such as ethanol, from sugarcane. Knowledge of the key transporters involved in sugar exchange between the vacuole and cytoplasm is thus relevant in the context of bioenergy (Grennan and Gragg, 2009).To facilitate the exchange of sugars across the tonoplast, plant vacuoles are equipped with a multitude of transporters (Neuhaus, 2007; Etxeberria et al., 2012; Martinoia et al., 2012) comprising both facilitated diffusion and active transport systems of vacuolar sugars (Martinoia et al., 2000). Typically, Suc is actively imported into vacuoles by tonoplast monosaccharide transporter (AtTMT1/AtTMT2; Schulz et al., 2011) and exported by the SUT4 family (AtSUC4, OsSUT2; Eom et al., 2011; Payyavula et al., 2011; Schulz et al., 2011). Two H⁺-dependent sugar antiporters, the vacuolar Glc transporter (AtVGT1; Aluri and Büttner, 2007) and AtTMT1 (Wormit et al., 2006), mediate Glc uptake across the tonoplast to promote carbohydrate accumulation in Arabidopsis (Arabidopsis thaliana). The Early Responsive to Dehydration-Like6 protein has been shown to export vacuolar Glc into the cytosol (Poschet et al., 2011), likely via an energy-independent diffusion mechanism (Yamada et al., 2010). Defects in these vacuolar sugar transporters alter carbohydrate partitioning and allocation and inhibit plant growth and seed yield (Aluri and Büttner, 2007; Wingenter et al., 2010; Eom et al., 2011; Poschet et al., 2011).In contrast to numerous studies on vacuolar transport of Suc and Glc, limited efforts have been devoted to the molecular mechanism of vacuolar Fru transport even though Fru is predominantly located in vacuoles (Martinoia et al., 1987; Voitsekhovskaja et al., 2006; Tohge et al., 2011). Vacuolar Fru is important for the regulation of turgor pressure (Pontis, 1989), antioxidative defense (Bogdanović et al., 2008), and signal transduction during early seedling development (Cho and Yoo, 2011; Li et al., 2011). Thus, control of Fru transport across the tonoplast is thought to be important for plant growth and development. One vacuolar Glc transporter from the Arabidopsis monosaccharide transporter family, VGT1, has been reported to mediate low-affinity Fru uptake when expressed in yeast (Saccharomyces cerevisiae) vacuoles (Aluri and Büttner, 2007). Yet, the high vacuolar uptake activity to Fru intimates the existence of additional high-capacity Fru-specific vacuolar transporters (Thom et al., 1982). Recently, quantitative mapping of a quantitative trait locus for Fru content of leaves led to the identification of the Fru-specific vacuolar transporter SWEET17 (Chardon et al., 2013).SWEET17 belongs to the recently identified SWEET (PFAM:PF03083) super family, which contains 17 members in Arabidopsis and 21 in rice (Oryza sativa; Chen et al., 2010; Frommer et al., 2013; Xuan et al., 2013). Based on homology with 27% to 80% amino acid identity, plant SWEET proteins were grouped into four subclades (Chen et al., 2010). Analysis of GFP fusions indicated that most SWEET transporters are plasma membrane localized. Transport assays using radiotracers in Xenopus laevis oocytes and sugar nanosensors in mammalian cells showed that they function as largely pH-independent low-affinity uniporters with both uptake and efflux activity (Chen et al., 2010, 2012). In particular, clade I and II SWEETs transport monosaccharides and clade III SWEETs transport disaccharides, mainly Suc (Chen et al., 2010, 2012). Mutant phenotypes and developmental expression of several SWEET transporters support important roles in sugar translocation between organs. The clade III SWEETs, in particular SWEET11 and 12, mediate the key step of Suc efflux from phloem parenchyma cells for phloem translocation (Chen et al., 2012). Moreover, SWEETs are coopted by pathogens, likely to provide energy resources and carbon at the site of infection (Chen et al., 2010). Mutations of SWEET8/Ruptured pollen grain1 in Arabidopsis, and RNA inhibition of OsSWEET11 (also called Os8N3 or Xa13) in rice, and petunia (Petunia hybrida) NEC1 resulted in male sterility (Ge et al., 2001; Yang et al., 2006; Guan et al., 2008), possibly caused by inhibiting the Glc supply to developing pollen (Guan et al., 2008). Interestingly, two members, SWEET16 and SWEET17, of the family localize to the tonoplast (Chardon et al., 2013; Klemens et al., 2013). Allelic variation or mutations that affect SWEET17 expression caused Fru accumulation in Arabidopsis leaves, indicating that it plays a key role in exporting Fru from leaf vacuoles (Chardon et al., 2013). A more recent study demonstrated that SWEET16 also functions as a vacuolar sugar transporter (Klemens et al., 2013). Surprisingly, however, SWEET17 expression in mature leaves was comparatively low (Chardon et al., 2013), which leads us to ask whether SWEET17 could mainly function in other tissues under specific developmental or environmental conditions. Although Arabidopsis SWEET17 has been shown to transport Fru in a heterologous system where it accumulated in part at the plasma membrane (Chardon et al., 2013), the biochemical properties of SWEET17 were still elusive. SWEET16 and SWEET17 from Arabidopsis belong to the clade IV SWEETs. Whether clade IV proteins both transport vacuolar sugars in planta deserves further studies.Here, we used GUS/GFP fusions to reveal the root-dominant expression and vacuolar localization of the SWEET17 protein in vivo and its regulation by Fru levels. Phenotypes of mutants and overexpressors were consistent with a role of SWEET17 in bidirectional Fru transport across root vacuoles. The uniport feature of SWEET17 transport was further confirmed using isolated mesophyll vacuoles. Similarly, SWEET16 is also shown to function in vacuolar sugar transport in roots. Our work, performed in parallel to the two other studies (Chardon et al., 2013; Klemens et al., 2013), provides direct evidence for Fru uniport by SWEET17 and presents functional analyses to uncover important roles of these vacuolar transporters in maintaining intracellular Fru homeostasis in roots.     

Isolation and Comparison of Eight SWEET17 Genes from Six Loquat Cultivars

Russian Journal of Plant Physiology - The plant SWEET (Sugars Will Eventually be Exported Transporters) proteins, as newly discovered sugar transporters, play various important roles during plant...     

Phytophthora sojae apoplastic effector AEP1 mediates sugar uptake by mutarotation of extracellular aldose and is recognized as a MAMP

Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex plant–Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography–mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the β-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity.

Phytophthora sojae apoplastic effector AEP1 triggers pattern-triggered immunity in nonhost plants and contributes to P. sojae virulence by promoting the uptake of extracellular sugar.     

Pseudozyma aphidis induces ethylene-independent resistance in plants

Species of the epiphytic fungus Pseudozyma are not pathogenic to plants and can be used as biocontrol agents against plant pathogens. Deciphering how they induce plant defense might contribute to their use for plant protection and expand our understanding of molecular plant–pathogen interactions. Here we show that Pseudozyma aphidis isolate L12, which is known to induce jasmonic acid- and salicylic acid-independent systemic resistance, can also activate local and systemic resistance in an ethylene-independent manner. We also show that P. aphidis localizes exclusively to the surface of the plant leaf and does not penetrate the mesophyll cells of treated leaves. We thus propose that P. aphidis acts via several mechanisms, and is an excellent candidate biocontrol agent.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.     

Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

Consolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.     

Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

Endoplasmic reticulum–plasma membrane contact sites (ER–PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER–PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER–PM tether that also functions in maintaining PM integrity. The ER–PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Arabidopsis synaptotagmins 1 and 3 are localized at endoplasmic reticulum–plasma membrane contact sites and during abiotic stress episodes control diacylglycerol homeostasis at the plasma membrane