To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana , laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host.

Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl-and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl-and chlorothiazolylneonicotinoids induce SA responses associated with enhanced stress tolerance.N eonicotinoids are the newest of the three major classes of insecticides, which also include the organophosphorus compounds and pyrethroids. Imidacloprid (IMI), with a chloropyridinyl (Cl-pyr) substituent, is the most important neonicotinoid, used primarily as a systemic compound absorbed and translocated by plants to control sucking insect pests (1). The neonicotinoids clothianidin (2) (CLO) and a metabolic precursor, the oxadiazine compound thiamethoxam (3, 4), which have chlorothiazolyl (Cl-thia) substituents, are also extensively used as systemic insecticides in plants. The neonicotinoids IMI and CLO are oxidatively cleaved in planta to 6-chloropyridinyl-3-carboxylic acid (CPA) and 2-chlorothiazolyl-5-carboxylic acid (CTA), respectively, among other metabolites (5). In studying metabolism of neonicotinoids in spinach (5) under insect-free conditions, we sometimes observed enhancement of foliage growth, plant vigor, and drought-tolerance. These remarkable effects of neonicotinoids directly on plants, independent of controlling insect pests, have also been noted by many researchers and farmers and documented in both research publications and patent disclosures, especially for IMI (6-8) and the CLO precursor, thiamethoxam (9). In addition, treatment with IMI...

The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant. A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates. By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides. Sucrose induced inward currents with a K 0.5 of 1.44 mM at pH 5 and a membrane potential of ؊137 mV. Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis. These glucosides were maltose, arbutin (hydroquinone-␤-D-glucoside), salicin (2-(hydroxymethyl)phenyl-␤-D-glucoside), ␣-phenylglucoside, ␤-phenylglucoside, ␣-paranitrophenylglucoside, ␤-paranitrophenylglucoside, and paranitrophenyl-␤-thioglucoside. In addition, turanose and ␣-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2. The results indicate that AtSUC2 is not highly selective for ␣-over ␤-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.Plant sucrose transporters (SUTs, also named SUCs) 1 are integral membrane proteins within the glycoside-pentosidehexuronide cation symporter family. SUTs are expressed in the plasma membrane of companion cells and sieve elements within the phloem, a specialized tissue within the plant vasculature, and catalyze the H ϩ -coupled uptake of sucrose. Sucrose is the primary photosynthetic product that is transported by the phloem to heterotrophic tissues such as roots, developing leaves, and seeds (1, 2). The Arabidopsis genome encodes nine SUT homologs (3). One of these, AtSUC2 (4), functions as the main phloem-loading transporter. AtSUC2 is highly expressed in the companion cells of source (photosynthetic) leaves (5) and is essential for the long distance transport of sucrose as evidenced by the severe phenotype of insertional mutants (6). As demonstrated by the [ 14 C]sucrose uptake of yeast expressing AtSUC2, the transporter has a high affinity for sucrose (K m ϭ 0.8 mM) (4) compared with Arabidopsis homologs AtSUT4 (7) and AtSUT2 (8).In plant cell protoplasts or plasma membrane vesicles, sucrose uptake is inhibited by a variety of glucosides (9, 10) providing insight into the structural requirements for binding to sucrose transporters. Results indicated that none of the fructosyl hydroxyls of sucrose interact specifically with the transporter (9). The ability of phenylthioglucoside to inhibit sucrose uptake and to serve as a transported substrate further indicated that the fructosyl moiety of sucrose presents a hydrophobic surface required for binding (9, 10). Substitutions of the glucosyl hydroxyls 3, 4, and 6 of ...

Oligonucleotide microarrays corresponding to over 16,000 genes were used to analyze changes in transcript accumulation in root tips of the Al-sensitive Medicago truncatula cultivar Jemalong genotype A17 in response to Al treatment. Out of 2,782 genes with significant changes in transcript accumulation, 324 genes were up-regulated and 267 genes were down-regulated at least twofold by Al. Up-regulated genes were enriched in transcripts involved in cell-wall modification and abiotic and biotic stress responses while down-regulated genes were enriched in transcripts involved in primary metabolism, secondary metabolism, protein synthesis and processing, and the cell cycle. Known markers of Al-induced gene expression including genes associated with oxidative stress and cell wall stiffening were differentially regulated in this study. Transcript profiling identified novel genes associated with processes involved in Al toxicity including cell wall modification, cell cycle arrest and ethylene production. Novel genes potentially associated with Al resistance and tolerance in M. truncatula including organic acid transporters, cell wall loosening enzymes, Ca(2+) homeostasis maintaining genes, and Al-binding were also identified. In addition, expression analysis of nine genes in the mature regions of the root revealed that Al-induced gene expression in these regions may play a role in Al tolerance. Finally, interfering RNA-induced silencing of two Al-induced genes, pectin acetylesterase and annexin, in A17 hairy roots slightly increased the sensitivity of A17 to Al suggesting that these genes may play a role in Al resistance.

Plant sugar will eventually be exported transporter (SWEET) sugar transporters have been implicated in various developmental processes where sugar efflux is essential, including sucrose loading of phloem for long-distance sugar transport, nectar secretion, embryo and pollen nutrition, and maintenance of sugar homeostasis in plant organs. Notably, these transporters are selectively targeted by pathogens to gain access to host sugars. In most cases, when SWEET function is blocked, the growth and virulence of the pathogen is also reduced. There is growing evidence to suggest that the lifestyle of the pathogen may dictate which SWEET or set of SWEET genes are recruited for pathogen growth and proliferation. Furthermore, SWEET transporters may also play a role in abiotic stress tolerance by enabling plant growth under unfavorable environmental conditions. This review provides an overview of the diverse functions of SWEET proteins in plant development, pathogen nutrition, and abiotic stress tolerance. In addition, utility of the model legume Medicago truncatula as a tool to elucidate SWEET function in diverse host-microbe interactions is discussed. V C 2015 IUBMB Life, 67(7): [461][462][463][464][465][466][467][468][469][470][471] 2015

In plants, the activation of immunity is often inversely correlated with growth. Mechanisms that control plant growth in the context of pathogen challenge and immunity are unclear. Investigating Arabidopsis infection with the powdery mildew fungus, we find that the Arabidopsis atypical E2F DEL1, a transcriptional repressor known to promote cell proliferation, represses accumulation of the hormone salicylic acid (SA), an established regulator of plant immunity. DEL1-deficient plants are more resistant to pathogens and slightly smaller than wild-type. The resistance and size phenotypes of DEL1-deficient plants are due to the induction of SA and activation of immunity in the absence of pathogen challenge. Moreover, Enhanced Disease Susceptibility 5 (EDS5), a SA transporter required for elevated SA and immunity, is a direct repressed target of DEL1. Together, these findings indicate that DEL1 control of SA levels contributes to regulating the balance between growth and immunity in developing leaves.

Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wildtype and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T 2 statistic). Functional analyses of T 2 -selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.

Background: Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn.