Maize hybrids derived from GM positive and negative segregant inbreds are compositionally equivalent: any observed differences are associated with conventional backcrossing practices.

In this study, we show that compositional differences in grain harvested from genetically modified (GM) maize hybrids derived from near-isogenic trait-positive and trait-negative segregant inbreds are more likely related to backcrossing practices than to the GM trait. To demonstrate this, four paired GM trait-positive (NK603: herbicide tolerance) and trait-negative near-isogenic inbred male lines were generated. These were crossed with two different females (testers) to create a series of trait-positive and trait-negative hybrid variants. The hypothesis was, that compositional variation within the hybrid variants would reflect differences associated with backcrossing practices and provide context to any observed differences between GM and non-GM hybrids. The F1 hybrids, as well as corresponding conventional comparator hybrids, were grown concurrently at four field sites across the United States during the 2013 season. Grain was harvested for compositional analysis; proximates (protein, starch, and oil), amino acids, fatty acids, minerals, tocopherols (α-, δ-, γ-), ß-carotene, phytic acid, and raffinose were measured. Statistical analysis showed that within each hybrid tester set, there were very few significant (p < 0.05) differences between the paired trait-positive and trait-negative hybrids or between the conventional comparators and the trait-positive or trait-negative hybrids. Assessments of the magnitudes of differences and variance component analysis highlighted that growing location, and the tester used in hybrid formation, had a markedly greater effect on composition than did the GM trait. Significantly, for each tester set, compositional differences within the trait-positive and trait-negative hybrid variants were greater than differences between the GM and non-GM comparators. Overall, GM trait insertion is not intrinsically a meaningful contributor to compositional variation, and observed differences between GM and non-GM comparators typically reflect incidental changes associated with conventional breeding practices. These results contribute to ongoing discussions on the relevance of negative segregants as comparators in GM assessments.

The haustorial transcriptomes of Uromyces appendiculatus and Phakopsora pachyrhizi and their candidate effector families.

Haustoria of biotrophic rust fungi are responsible for the uptake of nutrients from their hosts and for the production of secreted proteins, known as effectors, which modulate the host immune system. The identification of the transcriptome of haustoria and an understanding of the functions of expressed genes therefore hold essential keys for the elucidation of fungus-plant interactions and the development of novel fungal control strategies. Here, we purified haustoria from infected leaves and used 454 sequencing to examine the haustorial transcriptomes of Phakopsora pachyrhizi and Uromyces appendiculatus, the causal agents of soybean rust and common bean rust, respectively. These pathogens cause extensive yield losses in their respective legume crop hosts. A series of analyses were used to annotate expressed sequences, including transposable elements and viruses, to predict secreted proteins from the assembled sequences and to identify families of candidate effectors. This work provides a foundation for the comparative analysis of haustorial gene expression with further insights into physiology and effector evolution.

Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway.

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.

Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate with fungal penetration and haustoria formation.

Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.

Identification and analyses of candidate genes for rpp4-mediated resistance to Asian soybean rust in soybean.

Asian soybean rust is a formidable threat to soybean (Glycine max) production in many areas of the world, including the United States. Only five sources of resistance have been identified (Resistance to Phakopsora pachyrhizi1 [Rpp1], Rpp2, Rpp3, Rpp4, and Rpp5). Rpp4 was previously identified in the resistant genotype PI459025B and mapped within 2 centimorgans of Satt288 on soybean chromosome 18 (linkage group G). Using simple sequence repeat markers, we developed a bacterial artificial chromosome contig for the Rpp4 locus in the susceptible cv Williams82 (Wm82). Sequencing within this region identified three Rpp4 candidate disease resistance genes (Rpp4C1-Rpp4C3 [Wm82]) with greatest similarity to the lettuce (Lactuca sativa) RGC2 family of coiled coil-nucleotide binding site-leucine rich repeat disease resistance genes. Constructs containing regions of the Wm82 Rpp4 candidate genes were used for virus-induced gene silencing experiments to silence resistance in PI459025B, confirming that orthologous genes confer resistance. Using primers developed from conserved sequences in the Wm82 Rpp4 candidate genes, we identified five Rpp4 candidate genes (Rpp4C1-Rpp4C5 [PI459025B]) from the resistant genotype. Additional markers developed from the Wm82 Rpp4 bacterial artificial chromosome contig further defined the region containing Rpp4 and eliminated Rpp4C1 (PI459025B) and Rpp4C3 (PI459025B) as candidate genes. Sequencing of reverse transcription-polymerase chain reaction products revealed that Rpp4C4 (PI459025B) was highly expressed in the resistant genotype, while expression of the other candidate genes was nearly undetectable. These data support Rpp4C4 (PI459025B) as the single candidate gene for Rpp4-mediated resistance to Asian soybean rust.

Distinct biphasic mRNA changes in response to Asian soybean rust infection.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under water-limiting conditions.

Biofilms exist in a variety of habitats that are routinely or periodically not saturated with water, and residents must integrate cues on water abundance (matric stress) or osmolarity (solute stress) into lifestyle strategies. Here we examine this hypothesis by assessing the extent to which alginate production by Pseudomonas putida strain mt-2 and by other fluorescent pseudomonads occurs in response to water limitations and how the presence of alginate in turn influences biofilm development and stress tolerance. Total exopolysaccharide (EPS) and alginate production increased with increasing matric, but not solute, stress severity, and alginate was a significant component, but not the major component, of EPS. Alginate influenced biofilm architecture, resulting in biofilms that were taller, covered less surface area, and had a thicker EPS layer at the air interface than those formed by an mt-2 algD mutant under water-limiting conditions, properties that could contribute to less evaporative water loss. We examined this possibility and show that alginate reduces the extent of water loss from biofilm residents by using a biosensor to quantify the water potential of individual cells and by measuring the extent of dehydration-mediated changes in fatty acid composition following a matric or solute stress shock. Alginate deficiency decreased survival of desiccation not only by P. putida but also by Pseudomonas aeruginosa PAO1 and Pseudomonas syringae pv. syringae B728a. Our findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemical properties. Collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.

Cell envelope components contributing to biofilm growth and survival of Pseudomonas putida in low-water-content habitats.

Bacteria in terrestrial habitats frequently reside as biofilm communities on surfaces that are unsaturated, i.e. biofilms are covered in water films varying in thickness depending on the environmental conditions. Water availability in these habitats is influenced by the osmolarity of the water (solute stress) and by cellular dehydration imposed by matric stress, which increases as water content decreases. Unfortunately, we understand relatively little about the molecular mechanisms required for bacterial growth in low-water-content habitats. Here, we describe the use of mini-Tn5-'phoA to identify genes in Pseudomonas putida that are matric water stress controlled and to generate mutants defective in desiccation tolerance. We identified 20 genes that were induced by a matric stress but not by a thermodynamically equivalent solute stress, 11 genes were induced by both a matric and a solute stress, three genes were induced by a solute stress and three genes were repressed by a matric stress. Their patterns of expression were analysed in laboratory media, and their contribution to desiccation tolerance was evaluated. Twenty-six genes were homologous to sequences present in the completed P. putida KT2440 genome sequence or plasmid pWWO sequence that are involved in protein fate, nutrient or solute acquisition, energy generation, motility, alginate biosynthesis or cell envelope structure, and the function of five could not be predicted from the sequence. Together, these genes and their importance to desiccation tolerance provide a view of the environment perceived by bacteria in low-water-content habitats, and suggest that the mechanisms for adaptation for growth in low-water-content habitats are different from those for growth in high-osmolarity habitats.