Chiliang Chen

Current management of sudden death syndrome (SDS) of soybean, caused by Fusarium virguliforme, focuses on planting resistant varieties and improving soil drainage; however, these measures are not completely effective. A 6-year study evaluated the effects of cropping system diversification on SDS and soybean yield. SDS, root health, yield, and F. virguliforme density in soil were assessed in a naturally infested field trial comparing a 2-year cropping system consisting of a corn-soybean rotation and synthetic fertilizer applications with 3- and 4-year cropping systems consisting of corn-soybean-oat + red clover and corn-soybean-oat +alfalfa-alfalfa rotations, respectively, with both manure and low synthetic fertilizer rates. In 5 of 6 years, SDS incidence and severity were lower and yield higher in the 3- and 4-year systems than in the 2-year system. SDS severity and incidence were up to 17-fold lower in the diversified systems than in the 2-year system. Incidence and severity of SDS explained 45 to 87% of the variation in yield. Plants in the 2-year system generally showed more severe root rot and lower plant weights than plants in the diversified systems. F. virguliforme density in soil was up to fivefold greater in the 2-year system compared with the 4-year system. The processes responsible for the suppression of SDS and yield protection in the diversified cropping systems still need to be determined.

Summary: Arsenic, a highly cytotoxic and cancerogenic metalloid, is brought into the biosphere through geochemical sources and anthropogenic activities. A global biogeochemical arsenic biotransformation cycle exists in which inorganic arsenic species are transformed into organoarsenicals, which are subsequently mineralized again into inorganic arsenic compounds. Microorganisms contribute to this biotransformation process greatly and one of the organoarsenicals synthesized and degraded in this cycle is arsenobetaine. Its nitrogen‐containing homologue glycine betaine is probably the most frequently used compatible solute on Earth. Arsenobetaine is found in marine and terrestrial habitats and even in deep‐sea hydrothermal vent ecosystems. Despite its ubiquitous occurrence, the biological function of arsenobetaine has not been comprehensively addressed. Using Bacillus subtilis as a well‐understood platform for the study of microbial osmostress adjustment systems, we ascribe here to arsenobetaine both a protective function against high osmolarity and a cytoprotective role against extremes in low and high growth temperatures. We define a biosynthetic route for arsenobetaine from the precursor arsenocholine that relies on enzymes and genetic regulatory circuits for glycine betaine formation from choline, identify the uptake systems for arsenobetaine and arsenocholine, and describe crystal structures of ligand‐binding proteins from the OpuA and OpuB ABC transporters complexed with either arsenobetaine or arsenocholine.

Summary: The ABC‐transporters OpuB and OpuC fromBacillus subtilisfunction as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC‐transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate‐binding proteins between the correspondingopuBandopuCoperons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC‐mediated compatible solute import through transcriptional up‐regulation of the hybridopuB::opuCCoperon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand‐binding protein.

Moderate resolution imaging spectroradiometer (MODIS) satellite imagery from 2004 to 2013 were used to assess the field-specific risks of soybean sudden death syndrome (SDS) caused by Fusarium virguliforme in Iowa. Fields with a high frequency of significant decrease (>10%) of the normalized difference vegetation index (NDVI) observed in late July to middle August on historical imagery were hypothetically considered as high SDS risk. These high-risk fields had higher slopes and shorter distances to flowlines, e.g., creeks and drainages, particularly in the Des Moines lobe. Field data in 2014 showed a significantly higher SDS level in the high-risk fields than fields selected without considering NDVI information. On average, low-risk fields had 10 times lower F. virguliforme soil density, determined by quantitative polymerase chain reaction, compared with other surveyed fields. Ordinal logistic regression identified positive correlations between SDS and slope, June NDVI, and May maximum temperature, but high June maximum temperature hindered SDS. A modeled SDS risk map showed a clear trend of potential disease occurrences across Iowa. Landsat imagery was analyzed similarly, to discuss the ability to utilize higher spatial resolution data. The results demonstrated the great potential of both MODIS and Landsat imagery for SDS field-specific risk assessment.

Summary: Dimethylsulfoniopropionate ( DMSP ) is an abundant osmolyte and anti‐stress compound produced primarily in marine ecosystems. After its release into the environment, microorganisms can exploit DMSP as a source of sulfur and carbon, or accumulate it as an osmoprotectant. However, import systems for this ecophysiologically important compatible solute, and its stress‐protective properties for microorganisms that do not produce it are insufficiently understood. Here we address these questions using a well‐characterized set of B acillus subtilis mutants to chemically profile the influence of DMSP import on stress resistance, the osmostress‐adaptive proline pool and on osmotically controlled gene expression. We included in this study the naturally occurring selenium analogue of DMSP , dimethylseleniopropionate ( DMSeP ), as well as a set of synthetic DMSP derivatives. We found that DMSP is not a nutrient for B . subtilis , but it serves as an excellent stress protectant against challenges conferred by sustained high salinity or lasting extremes in both low and high growth temperatures. DMSeP and synthetic DMSP derivatives retain part of these stress protective attributes, but DMSP is clearly the more effective stress protectant. We identified the promiscuous and widely distributed ABC transporter OpuC as a high‐affinity uptake system not only for DMSP , but also for its natural and synthetic derivatives.

The foliar pathogenPseudomonas syringaeis a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of twoP. syringaestrains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible soluteN-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction ofglnA1expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.

Summary: The quaternary ammonium compound (QAC) choline is a major component of membrane lipids in eukaryotes and, if available to microbial colonists of plants, could provide benefits for growth and protection from stress. Free choline is found in homogenized plant tissues, but its subcellular location and availability to plant microbes are not known. Whole‐cell bacterial bioreporters of the phytopathogen Pseudomonas syringae were constructed that couple a QAC‐responsive transcriptional fusion with well‐characterized bacterial QAC transporters. These bioreporters demonstrated the presence of abundant free choline compounds released from germinating seeds and seedlings of the bean Phaseolus vulgaris, and a smaller but consistently detectable amount of QACs, probably choline, from leaves. The localization of P. syringae bioreporter cells to the surface and intercellular sites of plant tissues demonstrated the extracellular location of these QAC pools. Moreover, P. syringae mutants that were deficient in the uptake of choline compounds exhibited reduced fitness on leaves, highlighting the importance of extracellular choline to P. syringae on leaves. Our data support a model in which this choline pool is derived from the phospholipid phosphatidylcholine through plant‐encoded phospholipases that release choline into the intercellular spaces of plant tissues, such as for membrane lipid recycling. The consequent extracellular release of choline compounds enables their interception and exploitation by plant‐associated microbes, and thus provides a selective advantage for microbes such as P. syringae that are adapted to maximally exploit choline.

Pseudomonas aeruginosauses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline inP. aeruginosaphysiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, inP. aeruginosagrowth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1and ΔbetT1ΔbetT3mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline.betT1andbetT3expression was regulated by the repressor BetI and choline, whereascbcXWVexpression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediatedcbcXWVinduction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake inP. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.

Summary: Surprisingly little is known of the trehalose biosynthetic pathways in pseudomonads, despite the importance of trehalose to protecting cells from environmental stresses such as low water availability. The genome of the foliar pathogen Pseudomonas syringae pv. tomato strain DC3000 contains genes for two trehalose biosynthetic pathways, TreS and TreYZ, and lacks genes for the more common OtsAB pathway. Deletion of either the treS (PSPTO_2760‐2762) or treY/treZ (PSPTO_3125‐3134) locus eliminated trehalose accumulation and reduced bacterial growth under hyperosmotic conditions. In evaluating the role of trehalose in P. syringae fitness on leaves, we found that a double deletion mutant lacking these loci exhibited poorer survival than the wild type on tomato leaves over a 2‐week period in a growth chamber. Similarly, this mutant exhibited reduced survival on leaves of susceptible and resistant cultivars of the host plant tomato and of the non‐host plant soybean over a 10‐day period in field plots. Thus, the trehalose biosynthetic loci in P. syringae , which are highly conserved among pseudomonads, contributed to DC3000 fitness on leaves, supporting a role for trehalose in P. syringae survival and population maintenance in the phyllosphere.