Joseph C. Kuhl

A Unique Set of 11,008 Onion Expressed Sequence Tags Reveals Expressed Sequence and Genomic Differences between the Monocot Orders Asparagales and Poales

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.

Characterization and mapping of Rpi1, a late-blight resistance locus from diploid (1EBN) Mexican Solanum pinnatisectum

Abstract. Solanum is a diverse genus with over 200 species occupying a range of habitats from the Southwestern United States to Central Chile. Germplasm evaluations have focused on species that can be crossed with S. tuberosum, while Mexican diploid (2n=2x=24) Solanum species with an Endosperm Balance Number (EBN) of 1 have received less attention because of poor crossability due to their ploidy and EBN. Recent changes in Phytophthora infestans populations have increased the need for new sources of genetic resistance to this fungus. We have characterized resistance to P. infestans in the Mexican 2x(1EBN) species S. pinnatisectum. An interspecific hybrid between resistant S. pinnatisectum and susceptible S. cardiophyllum plants was backcrossed to S. cardiophyllum to generate a family segregating for late-blight resistance. The diploid (1EBN) genetic map generated with 99 RFLP markers revealed extensive synteny with previously published potato maps. A single dominant late-blight resistance locus (Rpi1) from S. pinnatisectum was mapped to chromosome 7, a region previously not associated with late-blight resistance. Characterization of the P. infestans isolate used for disease evaluations revealed that it possessed the avirulence gene corresponding to the R9 resistance locus, indicating that Rpi1 could possibly correspond to R9.

CRT1, an Arabidopsis ATPase that Interacts with Diverse Resistance Proteins and Modulates Disease Resistance to Turnip Crinkle Virus

Plant immunity frequently involves the recognition of pathogen-encoded avirulence (avr) factors by their corresponding plant resistance (R) proteins. This triggers the hypersensitive response (HR) where necrotic lesions formed at the site(s) of infection help restrict pathogen spread. HRT is an Arabidopsis R protein required for resistance to turnip crinkle virus (TCV). In a genetic screen for mutants compromised in the recognition of TCVs avr factor, we identified crt1 (compromised recognition of TCV), a mutant that prematurely terminates an ATPase protein. Following TCV infection, crt1 developed a spreading HR and failed to control viral replication and spread. crt1 also suppressed HR-like cell death induced by ssi4, a constitutively active R protein, and by Pseudomonas syringae carrying avrRpt2. Furthermore, CRT1 interacts with HRT, SSI4, and two other R proteins, RPS2 and Rx. These data identify CRT1 as an important mediator of defense signaling triggered by distinct classes of R proteins.

Differential effects of environment on potato phenylpropanoid and carotenoid expression

BackgroundPlant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida).ResultsPhenylpropanoids, including chlorogenic acid (CGA), were higher in samples from the northern latitudes, as was the expression of phenylpropanoid genes including phenylalanine ammonia lyase (PAL), which had over a ten-fold difference in relative abundance. Phenylpropanoid gene expression appeared coordinately regulated and was well correlated with metabolite pools, except for hydroxycinnamoyl-CoA:quinatehydroxcinnamoyl transferase (HQT; r = -0.24). In silico promoter analysis identified two cis-acting elements in the HQT promoter not found in the other phenylpropanoid genes. Anthocyanins were more abundant in Alaskan samples and correlated with flavonoid genes including DFR (r = 0.91), UFGT (r = 0.94) and F3H (r = 0.77). The most abundant anthocyanin was petunidin-3-coum-rutinoside-5-glu, which ranged from 4.7 mg g-1 in Alaska to 2.3 mg g-1 in Texas. Positive correlations between tuber sucrose and anthocyanins (r = 0.85), suggested a stimulatory effect of sucrose. Smaller variation was observed in total carotenoids, but marked differences occurred in individual carotenoids, which had over a ten-fold range. Violaxanthin, lutein or zeaxanthin were the predominant carotenoids in tubers from Alaska, Texas and Florida respectively. Unlike in the phenylpropanoid pathway, poor correlations occurred between carotenoid transcripts and metabolites.ConclusionAnalysis of tuber secondary metabolism showed interesting relationships among different metabolites in response to collective environmental influences, even under conditions that minimize stress. The variation in metabolites shows the considerable phenotypical plasticity possible with tuber secondary metabolism and raises questions about to what extent these pathways can be stimulated by environmental cues in a manner that optimizes tuber phytonutrient content while protecting yields. The differences in secondary metabolites may be sufficient to affect nutritional quality.

SlNAC1, a stress‐related transcription factor, is fine‐tuned on both the transcriptional and the post‐translational level

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.

Construction and expression of a synthetic wheat storage protein gene

A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E. coli. This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization. The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E. coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats. The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs. The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE.

Evidence of a Monogenic Nature of the Nz Gene Conferring Resistance Against Potato virus Y Strain Z (PVYZ) in Potato

Hypersensitive resistance (HR) to Potato virus Y (PVY) in potato (Solanum tuberosum) is conferred by strain-specific N genes. Two such genes have been identified in potato so far, Nytbr conferring HR to PVYO, and Nctbr conferring HR to PVYC. A third, putative gene Nztbr was proposed to confer HR against a distinct strain PVYZ. However, due to the scarcity of the PVYZ isolates of PVY, no formal proof of the monogenic nature of this new gene, Nztbr, was available until now. Here, we report on a genetic study of the Nztbr inheritance in three crosses between cultivars Maris Bard (Ny:Nz) and King Edward (ny:nz), and Maris Bard (Ny:Nz) and Russet Norkotah (ny:nz). A fully-sequenced PVYZ isolate, L26, was used to screen the parents and progeny for a virus-induced HR phenotype in foliage. Based on the phenotypic analysis of 203 progeny, segregation of HR phenotype in the PVYZ-infected plants was found to be 1:1, indicating a monogenic, dominant nature of the Nztbr gene. Since the PVYZ strain includes PVYNTN isolates associated with tuber necrotic ringspot disease (PTNRD) in susceptible potato cultivars, the Nztbr gene represents a valuable source of HR against PTNRD-inducing PVY isolates. This is the first demonstration that Nztbr is a single, dominant N gene in potato conferring resistance to the PVYZ-NTN strain.ResumenLa Resistencia hipersensible (HR) al virus Y de la papa (PVY) en papa (Solanum tuberosum) es conferida por genes N específicos por variante. Se han identificado dos de esos genes en papa hasta ahora, Nytbr, que confiere HR al PVYO, y Nctbr que confiere HR al PVYC. Un tercer supuesto gen Nztbr se propuso que confiere HR contra una variante distinta de PVYZ. No obstante, debido a lo esporádico del aislamiento de PVY tipo PVYZ, no estuvo disponible alguna prueba formal de la naturaleza monogénica de este nuevo gen, Nztbr, hasta ahora. Aquí reportamos un estudio genético de la heredabilidad de Nztbr en tres cruzas entre las variedades Maris Bard (Ny:Nz) y King Edward (ny:nz), y Maris Bard (Ny:Nz) y Russet Norkotah (ny:nz). Se utilizó un aislamiento completamente secuenciado de PVYZ, L26, para estudiar a los progenitores y a la progenie de un fenotipo con HR inducida por el virus en follaje. Con base en el análisis fenotípico de 203 progenies, se encontró que la segregación del fenotipo HR en las plantas infectadas con PVYZ fue de 1:1, indicando una naturaleza monogénica, dominante, del gen Nztbr. Debido a que la variante PVYZ incluye aislamientos de PVYNTN asociados con la enfermedad de la mancha anular necrótica del tubérculo (PTNRD) en variedades de papa susceptibles, el gen Nztbr representa una fuente valiosa de HR contra los aislamientos de PVY que inducen la PTNRD. Esta es la primera demostración que Nztbr es un solo gen N, dominante, en papa, que confiere resistencia a la variante PVYZ-NTN.

A morphological and molecular study in the Deschampsia cespitosa complex (Poaceae; Poeae; Airinae) in northern North America

PREMISE OF THE STUDY In the North American Arctic, the existence of one or several taxa closely related to Deschampsia cespitosa var. cespitosa has remained a puzzle for many years. Extreme morphological variation, lack of clear limits between alleged forms, and an extended geographic range often render identification keys incomplete, and raise the question of how many species this taxon represents. METHODS Morphological and molecular analysis, including multivariate statistics, ITS and the cpDNA marker trnK-rps16, was conducted on D. cespitosa var. cespitosa and related taxa using 201 herbarium specimens from northern North America (Alaska, Canada, and Greenland). Fifty-three morphological centeracters were recorded from all specimens, while sequences were retrieved from 167 specimens. KEY RESULTS Results show that Deschampsia cespitosa (L.) P. Beauv. var. cespitosa, D. cespitosa subsp. alpina (L.) Tzvelev, D. cespitosa subsp. beringensis (Hultén) W. E. Lawr., D. brevifolia R. Br., D. cespitosa (L.) P. Beauv. subsp. glauca (Hartm.) C. Hartm., D. mackenzieana Raup, D. cespitosa subsp. orientalis Hultén, and D. pumila (Griseb.) Ostenf. differed significantly in a few morphological variables, but molecularly are a closely related group with several sequences and haplotypes that are nearly identical. CONCLUSIONS Overall, the evidence points to the existence of a single species, Deschampsia cespitosa. The occurrence of slightly different morphological types related to specific geographical distributions allows recognition of three additional taxa at the infraspecific level, D. cespitosa subsp. alpina, D. cespitosa subsp. beringensis, and D. brevifolia. All studied taxa showed morphological variation in a gradient, suggesting the existence of phenotypic plasticity.

Plant programmed cell death caused by an autoactive form of Prf is suppressed by co-expression of the Prf LRR domain.

In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.

A genetic study of unilateral incompatibility between diploid (1EBN) Mexican species Solanum pinnatisectum and S. cardiophyllum subsp. cardiophyllum

Abstract. Many angiosperms have developed mechanisms to prevent self pollination and inbreeding. The most widespread system of self incompatibility is gametophytic, in which pollen tube growth is inhibited in the style by the action of a single locus (S-locus). Similar inhibition of pollen tube growth can also be observed in interspecific crosses, where successful pollinations occur in only one direction, termed unilateral incompatibility. We observed unilateral incompatibility in crosses between Solanum pinnatisectum and S. cardiophyllum, and viable interspecific hybrid seed was generated only when S. pinnatisectum was the pistillate parent. Segregations in the backcross to S. cardiophyllum revealed two independently segregating putative loci controlling interspecific crossability. Both putative loci were inherited independently of the S-locus. Segregation ratios in the backcross to the S.pinnatisectum parent did not agree with those observed in the backcross to the S. cardiophyllum parent. These reciprocal backcross families revealed that unilateral incompatibility is not conditioned by a single locus, and is inherited independently of the S-locus. Segregation disparity between the two backcross families may be due to distorted transfer of specific alleles or the presence of different mechanisms controlling pistil and pollen recognition systems in the two species.