Robert G. Upchurch

Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress

Stress acclimating plants respond to abiotic and biotic stress by remodeling membrane fluidity and by releasing α-linolenic (18:3) from membrane lipids. The modification of membrane fluidity is mediated by changes in unsaturated fatty acid levels, a function provided in part by the regulated activity of fatty acid desaturases. Adjustment of membrane fluidity maintains an environment suitable for the function of critical integral proteins during stress. α-Linolenic acid, released from membrane lipid by regulated lipase activity, is the precursor molecule for phyto-oxylipin biosynthesis. The modulation of chloroplast oleic acid (18:1) levels is central to the normal expression of defense responses to pathogens in Arabidopsis. Oleic (18:1) and linolenic (18:2) acid levels, in part, regulate development, seed colonization, and mycotoxin production by Aspergillus spp.

CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean.

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.

Identification and characterization of transcript polymorphisms in soybean lines varying in oil composition and content

BackgroundVariation in seed oil composition and content among soybean varieties is largely attributed to differences in transcript sequences and/or transcript accumulation of oil production related genes in seeds. Discovery and analysis of sequence and expression variations in these genes will accelerate soybean oil quality improvement.ResultsIn an effort to identify these variations, we sequenced the transcriptomes of soybean seeds from nine lines varying in oil composition and/or total oil content. Our results showed that 69,338 distinct transcripts from 32,885 annotated genes were expressed in seeds. A total of 8,037 transcript expression polymorphisms and 50,485 transcript sequence polymorphisms (48,792 SNPs and 1,693 small Indels) were identified among the lines. Effects of the transcript polymorphisms on their encoded protein sequences and functions were predicted. The studies also provided independent evidence that the lack of FAD2-1A gene activity and a non-synonymous SNP in the coding sequence of FAB2C caused elevated oleic acid and stearic acid levels in soybean lines M23 and FAM94-41, respectively.ConclusionsAs a proof-of-concept, we developed an integrated RNA-seq and bioinformatics approach to identify and functionally annotate transcript polymorphisms, and demonstrated its high effectiveness for discovery of genetic and transcript variations that result in altered oil quality traits. The collection of transcript polymorphisms coupled with their predicted functional effects will be a valuable asset for further discovery of genes, gene variants, and functional markers to improve soybean oil quality.

Transgenic assessment of CFP-mediated cercosporin export and resistance in a cercosporin-sensitive fungus.

Abstract. Cercosporin is a toxic polyketide produced by many phytopathogenic members of the fungal genus Cercospora. Cercospora species, themselves, exhibit the highest level of self-resistance to this almost universally toxic photosensitizer. Although the mechanism of cercosporin self-resistance is multi-faceted, partial resistance does appear to be provided by the encoded product of CFP (cercosporin facilitator protein), a gene recently isolated from the pathogen of soybean, C. kikuchii. CFP has significant similarity to the major facilitator superfamily of integral membrane transport proteins. We expressed CFP in the cercosporin non-producing, cercosporin-sensitive fungus, Cochliobolus heterostrophus, in order to assess the transport activity of CFP and the contribution of CFP to cercosporin resistance in a fungal species free of endogenous toxin production. Expression of the CFP transgene in this fungus results in increased resistance to cercosporin due, apparently, to its export out of the fungus.

Ergosterol as a Quantifiable Biomass Marker for Diaporthe phaseolorum and Cercospora kikuchii

The relationship between ergosterol content and biomass was determined for the soybean fungal pathogens Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. sojae, causal agent of Phomopsis seed decay, and Cercospora kikuchii (Matsumoto & Tomoy.), causal agent of leaf blight and purple seed stain. Biomass was manipulated by varying incubation period, and ergosterol was quantified by high-pressure liquid chromatography. Fungal dry mass was linearly correlated with ergosterol content (r2 = 0.90, P < 0.05 for D. phaseolorum, and r2 = 0.95, P < 0.01 for C. kikuchii). In vitro ergosterol content of fungi was 3.16 μg/mg for D. phaseolorum and 2.85 μg/mg for C. kikuchii. Ergosterol content of inoculated seed was qualitatively correlated with observed seed colonization by both pathogens. Soybean variety had a significant effect on fungal colonization by D. phaseolorum and ergosterol content. Results show that ergosterol content can be used to quantify colonization of soybean seed by both pathogens.

Expression of the cercosporin transporter, CFP, in tobacco reduces frog-eye lesion size

The cercosporin Major Facilitator Superfamily (MFS) transporter, CFP, under the control of the CaMV 35S promoter, was introduced into the Xanthi cultivar of tobacco by Agrobacterium-mediated transformation. CFP+ transgenic plants were physically indistinguishable from non-transgenic Xanthi and progressed normally through growth to seed set. Accumulation of CFP in the leaf membrane fraction of CFP+ transgenic plants was associated with decreased cercosporin phytotoxicity. Frog-eye leaf lesions on CFP+ transgenic plants infected with Cercospora nicotianae conidia were smaller but were similar in number to those on non-transgenic plants. We conclude that transgenic expression of CFP may have relevance for a disease control strategy in Cercospora-plant pathosystems where cercosporin is implicated in pathogen virulence.

Relationships Between Oleic and Linoleic Acid Content and Seed Colonization by Cercospora kikuchii and Diaporthe phaseolorum

Compared with standard cultivars, seed of mid-oleic soybean genotypes sometimes have shown increased colonization by Cercospora kikuchii in the field as judged by increased levels of purple-stained seed. To examine relationships between oleic and linoleic acid levels in soybean seed and postharvest seed colonization by two fungal seed pathogens, we inoculated seed with differing oleic:linoleic acid (O/L) ratios. Seed with defined O/L ratios were produced by allowing seed development of two isogenic soybean lines to occur in three different air temperature environments. Seed produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated with mycelium preparations of the fungal seed pathogens C. kikuchii or Diaporthe phaseolorum var. sojae. Fungal biomass of infected seed was quantified by measuring in vitro ergosterol content. For both soybean lines, colonization by C. kikuchii was positively correlated with the O/L ratio (r = 0.55, P < 0.03) and oleic acid content (r = 0.61, P < 0.02), and negatively correlated with linoleic (r = -0.60, P < 0.02) and linolenic (r = -0.58, P < 0.03) acid content. No association was found between the extent of seed colonization by D. phaseolorum and the seed O/L ratio. Our data suggest that the O/L ratio may be related to soybean seed colonization by C. kikuchii, but there is no evidence of a relationship with D. phaseolorum var. sojae colonization.

Electrophoretic karyotype of Cercospora kikuchii

Classical genetic analyses are not possible with the phytopathogenic fungus Cercospora kikuchii since no sexual stage has been identified. To facilitate gene mapping and to develop an understanding of the genome organization of C. kikuchii, an electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis (CHEF). Eight chromosomes, two of which migrate as a doublet, have been separated into seven bands ranging from 2.0 to 5.5 Mb. Using this determination of chromosome number and size, the total genome size of C. kikuchii is estimated to be 28.4 Mb. In addition, genes encoding tubulin, ribosomal DNA, and four previously isolated light-enhanced cDNAs from C. kikuchii were assigned to chromosomes by Southern-hybridization analysis of CHEF blots.

Effects of temperature during soybean seed development on defense-related gene expression and fungal pathogen accumulation.

Soybean [Glycine max (L.) Merr] plants were exposed to three temperature regimens during seed development to investigate the effect of temperature on the expression of eight defense-related genes and the accumulation of two fungal pathogens in inoculated seeds. In seeds prior to inoculation, either a day/night warm (34/26°C) or a cool temperature (22/18°C) relative to normal (26/22°C) resulted in altered patterns of gene expression including substantially lower expression of PR1, PR3 and PR10. After seed inoculation with Cercospora kikuchii, pathogen accumulation was lowest in seeds produced at 22/18°C in which of all defense genes, MMP2 was uniquely most highly induced. For seeds inoculated with Diaporthe phaseolorum, pathogen accumulation was lowest in seeds produced at 34/26°C in which of all defense genes, PR10 was uniquely most highly induced. Our detached seed assays clearly demonstrated that the temperature regimens we applied during seed development produced significant changes in seed defense-related gene expression both pre- and post inoculation and our findings support the hypothesis that global climate change may alter plant–pathogen interactions and thereby potentially crop productivity.

Genetic regulation of cercosporin production inCercospora kikuchii

The large and diverseCercospora genus of plant pathogenic fungi includes many species that are causal agents of economically relevant leaf, stem, and seed blights of numerous crop plants. Several of these pathogens produce the red, photoactivated, phytotoxic polyketide toxin cercosporin. This mycotoxin is a crucial pathogenicity factor in the development of leaf and pod blights by the seed-borne soybean fungal pathogenCercospora kikuchii. Although certain cultivars may be less susceptible to the leaf- and pod-infection phases of the fungus, there are no soybean cultivars with resistance to cercosporin. A newly isolated gene fromC. kikuchil, known as LE6, is essential for cercosporin production and pathogenicity. Therefore, genetic manipulation of this gene may affect resistance to cercosporin. Transcription of LE6 is regulated by light. The expression of cercosporin also may be inhibited by certain growth media and other natural products. Modification of cultivar screens that target LE6 may greatly enhance the possibility of finding native resistance to this soybean pathogen. Soybean germplasm that produces strong LE6 downregulating or inhibiting compounds may enhance pathogen resistance. Thus knowledge of the genetic and physiological regulation of cercosporin should provide new technological strategies for biocontrol of mycotoxins and the development of soybean breeding lines that exhibit durable resistance toC. kikuchii.