Gene B5 in Cotton Confers High and Broad Resistance to Bacterial Blight and Conditions High Amounts of Sesquiterpenoid Phytoalexins.

Bacterial blight resistance gene B5 has received little attention since it was first described in 1950. A near-isogenic line (NIL) of Gossypium hirsutum cotton, AcB5, was generated in an otherwise bacterial-blight-susceptible 'Acala 44' background. The introgressed locus B5 in AcB5 conferred strong and broad-spectrum resistance to bacterial blight. Segregation patterns of test crosses under Oklahoma field conditions indicated that AcB5 is likely homozygous for resistance at two loci with partial dominance gene action. In controlled-environment conditions, two of the four copies of B5 were required for effective resistance. Contrary to expectations of gene-for-gene theory, AcB5 conferred high resistance toward isogenic strains of Xanthomonas citri subsp. malvacearum carrying cloned avirulence genes avrB4, avrb7, avrBIn, avrB101, and avrB102, respectively, and weaker resistance toward the strain carrying cloned avrb6. The hypothesis that each B gene, in the absence of a polygenic complex, triggers sesquiterpenoid phytoalexin production was tested by measurement of cadalene and lacinilene phytoalexins during resistant responses in five NILs carrying different B genes, four other lines carrying multiple resistance genes, as well as susceptible Ac44E. Phytoalexin production was an obvious, but variable, response in all nine resistant lines. AcB5 accumulated an order of magnitude more of all four phytoalexins than any of the other resistant NILs. Its total levels were comparable to those detected in OK1.2, a highly resistant line that possesses several B genes in a polygenic background.

Pyramiding B genes in cotton achieves broader but not always higher resistance to bacterial blight.

Near-isogenic lines of upland cotton (Gossypium hirsutum) carrying single, race-specific genes B4, BIn, and b7 for resistance to bacterial blight were used to develop a pyramid of lines with all possible combinations of two and three genes to learn whether the pyramid could achieve broad and high resistance approaching that of L. A. Brinkerhoff's exceptional line Im216. Isogenic strains of Xanthomonas axonopodis pv. malvacearum carrying single avirulence (avr) genes were used to identify plants carrying specific resistance (B) genes. Under field conditions in north-central Oklahoma, pyramid lines exhibited broader resistance to individual races and, consequently, higher resistance to a race mixture. It was predicted that lines carrying two or three B genes would also exhibit higher resistance to race 1, which possesses many avr genes. Although some enhancements were observed, they did not approach the level of resistance of Im216. In a growth chamber, bacterial populations attained by race 1 in and on leaves of the pyramid lines decreased significantly with increasing number of B genes in only one of four experiments. The older lines, Im216 and AcHR, exhibited considerably lower bacterial populations than any of the one-, two-, or three-B-gene lines. A spreading collapse of spray-inoculated AcBIn and AcBInb7 leaves appears to be a defense response (conditioned by BIn) that is out of control.

Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya.

To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity.

2,7-Dihydroxycadalene and lacinilene C, sesquiterpenoid phytoalexins that accumulate at infection sites during the hypersensitive resistant response of cotton foliage to Xanthomonas campestris pv. malvacearum, have light-dependent toxicity toward host cells, as well as toward the bacterial pathogen. Adaxial epidermal cells surrounding and sometimes covering infection sites turn red. The red cells exhibited 3-4-fold higher absorption at the photoactivating wavelengths of sunlight than nearby colorless epidermal cells. Red epidermal cells protected underlying palisade mesophyll cells from the toxic effects of 2,7-dihydroxycadalene plus sunlight, indicating a role for epidermal pigments in protecting living cells that surround infection sites from toxic effects of the plant's own phytoalexins. A semi-quantitative survey of UV-absorbing substances extracted from epidermal strips from inoculated and mock-inoculated cotyledons indicated that the principal increase in capacity to absorb the photoactivating wavelengths was due to a red anthocyanin and a yellow flavonol, which were identified as cyanidin-3-O-beta-glucoside and quercetin-3-O-beta-glucoside, respectively.

Changes in homogalacturonans and enzymes degrading them during cotton cotyledon expansion.

Changes in homogalacturonans (HGs) and enzymes degrading them have been investigated during cotton (Gossypium hirsutum L.) cotyledon expansion. Using an in vivo assay for pectin-degrading enzymes that involves fluorescent labeled oligomers of GalA as substrate and capillary electrophoresis for product analysis, we found that endo- and exo-polygalacturonases are present in the cotyledon extracellular spaces, and there are dramatic changes in the levels of both activities as the cotyledons change their rate of expansion. Capacity for endo-polygalacturonase activity was highest during the initial stages of cotyledon expansion. However, for exo-polygalacturonase activity it was highest in the later stages of expansion. Cell walls were prepared from 3-, 5-, and 7-day-old cotton cotyledons and treated with liquid HF at -23 degrees C. This treatment cleaves the glycosidic linkages of most neutral sugars in the walls without degrading HGs. HGs with a relatively high degree of esterification can then be solubilized with water, and those with low esterification can be solubilized with concentrated imidazole buffer. The majority of HGs were obtained in the water extracts. The degrees of esterification were 57%, 47%, and 47% in water extracts and 34%, 25%, and 27% in imidazole extracts, in 3-, 5-, and 7-day-old cotton cotyledons, respectively. Using a PA100 ion-exchange column, the members of a GalA homologous series up to approximately 70 residues can be separated. The results from HG molecular length distribution analysis indicated that the HG at 3 days was on average shorter than that in the older cotyledons, perhaps reflecting the higher level of endo-polygalacturonase activity at this stage of more rapid growth.

A Technique for Precise Inoculation of the Internal Phyllosphere of Cotton with Xanthomonas campestris pv. malvacearum.

ABSTRACT A technique was developed to inoculate uniformly and gently the internal phyllosphere from the upper surface of cotton leaves with the phytopathogenic bacterium Xanthomonas campestris pv. malvacearum. The inoculum consisted of 2 to 3 x 10(7) CFU/ml in CaCO(3)-saturated sterile distilled water containing 0.02%, vol/vol, of the wetting agent Silwet L-77. A custom-made inoculation apparatus was employed to immerse a circular area of the adaxial surface of a leaf in inoculum for 90 s. This resulted in uniform, passive entry of bacteria into the substomatal chambers, producing an endophytic bacterial population of 2 x 10(4) CFU/cm(2). Microscopic signs of infection were visible 48 to 72 h after inoculation. In susceptible leaves, uniformly distributed water-soaked spots were observed 7 to 8 days after inoculation. When the technique was used on resistant leaves, the autofluorescence that is characteristic of hypersensitively necrotic cells developed in the guard cells and palisade cells lining substomatal chambers, but not in the underlying spongy mesophyll.

Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets.

The potential of DNA microarrays for detection of plant viruses is hampered by underutilization of sequence-independent amplification methods for target nucleic acid enrichment. A microarray system is described for an unbiased detection of plant viruses using both short (30 nt) and long (50 and 70 nt) oligonucleotide probes. The assay involves amplification of target nucleic acid using random primers followed by in vitro transcription whose cRNA product is labeled chemically, fragmented and used as target for hybridization. Initial optimization tests with Turnip vein clearing virus and Cauliflower mosaic virus showed increased hybridization efficiency with shorter cDNA targets (100 bp) and longer probes (50 and 70 nt). The system was validated in pure and mixed samples by detection of three Tymovirus species: Asclepias asymptomatic virus, Kennedya yellow mosaic virus and Turnip yellow mosaic virus. The method could detect sequence variants with 70-75% or higher sequence identity, indicating the possible utility of the approach for virus discovery. Array performance comparison of long probes demonstrated the competence of 50-mers to provide a satisfactory balance between detection sensitivity and specificity. The work described is a significant step towards a method to assess, in one assay, the presence of a large diversity of relatives of known viruses of plants.

The conceptual centrality of causal cycles.

How do causal cycles affect judgments of conceptual centrality? Generally, a feature is central to a concept to the extent that other features in the concept depend on it, thereby rendering it immutable from the concept (Sloman, Love, & Ahn, 1998). Previous research on conceptual centrality has focused primarily on features involved in four major types of dependency structures: simple cause-effect relations, causal chains, common-cause structures, and common-effect structures. Causal cycles are a fifth type of dependency structure commonly reported in people's real-life concepts, yet to date, they have been relatively ignored in research on conceptual centrality. The results of six experiments suggest that previously held assumptions about the conceptual representation of cycles are incorrect. We discuss the implications of these findings for our understanding of theory-based concepts.

Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons.

Rhamnogalacturonan lyase (RG lyase) activity has been detected and its relative activity measured in vivo during the expansion of cotton (Gossypium hirsutum L.) cotyledons. Rhamnogalacturonan (RG) oligomers labeled with a fluorescent tag were injected into the intercellular spaces of cotton cotyledons and, after incubation, the digested substrate was rinsed out. Enzyme digestion products were detected and identified by capillary zone electrophoresis. Rhamnogalacturonan lyase products were identified as such by co-migration with the digestion products of linear RG oligomers when the oligomers were treated with fungal RG lyase but not when treated with fungal RG hydrolase. In addition, reaction of plant RG lyase digestion products of RG oligomers with I(2)/KI, which selectively removes unsaturated galactopyranosyluronic acid (GaLap) residues formed at the non-reducing end of the oligomer, converted the plant digestion products into RG oligomers that co-migrated with fungal RG hydrolase products. The activity of the enzyme in the intercellular spaces of cotton cotyledons is very low and could be detected most easily when not >0.03 nmol of substrate was injected in a approximately 0.7-cm(2) area and incubated in vivo for 2-6 h. Rhamnogalacturonan lyase activity was the highest in rapidly expanding 3- to 4-day-old cotyledons and gradually decreased during the slow-down in expansion over the next 2-3 days. The RG lyase activity was also detected when the APTS (8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt)-labeled substrates were introduced into intercellular spaces by infiltration instead of injection, indicating that the activity was not induced by wounding or released into the apoplast by cell damage. An exo-RG galacturonohydrolase activity was also found, but RG hydrolase and exo-RG rhamnohydrolase were not detected.

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.

A global assembly of cotton ESTs.

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A(T) and D(T) genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics.