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.

Inhibitor and substrate activities of sesquiterpene olefins toward +-δ-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1).

Several lines of evidence indicate that (+)-δ-cadinene-8-hydroxylase (CYP706B1) plays an important role in biosynthesis of gossypol in Gossypium arboreum L. (Luo et al., 2001; Wang et al., 2003). The catalytically active enzyme has been expressed in yeast microsomes. Some microsomal preparations conjugated the hydroxylated (+)-δ-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-ß-d-glucoside (OG), a non-ionic detergent, (+)-δ-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-δ-cadinene. OG had little effect on K(m) and slightly stimulated apparent V(max). Enzymic activity was more than 10-fold more sensitive to inhibition by the N-substituted imidazole clotrimazole than to miconazole. Sesquiterpene olefins (-)-δ-cadinene, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and a mixture of (-)- and (+)-α-copaene were inhibitory to hydroxylation of (+)-δ-cadinene. In addition, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and, to a smaller extent, (-)-δ-cadinene served as alternative substrates for (+)-δ-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, α-humulene and α-copaene are found in lysigenous glands of cotton (Elzen et al., 1985), which are also the site of gossypol accumulation (Bell et al., 1978; Mace et al., 1976) and the probable site of its biosynthesis.

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.

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.

8-Hydroxy-(+)-delta-cadinene is a precursor to hemigossypol in Gossypium hirsutum.

[(3)H](+)-delta-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-(3)H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [(3)H](+)-8-hydroxy-delta-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [(3)H](+)-8-hydroxy-delta-cadinene had been converted to hemigossypol.

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.

Four near-isogenic lines of cotton with different genes for bacterial blight resistance.

ABSTRACT The development and genetic characterization of four near-isogenic lines (NILs) of cotton (Gossypium hirsutum) is described herein. Each line contains a single, but different, gene for resistance to bacterial blight caused by Xanthomonas campestris pv. malvacearum. The lines were derived using at least six backcrosses to the susceptible recurrent parent 'Acala 44', followed by single plant-progeny row selection for uniformity. The NILs are homozygous for the B(2), B(4), B(In), or b(7) genes and are designated as AcB(2), AcB(4), AcB(In), and Acb(7), respectively. In the 'Acala 44' background, B(2), B(4), and B(In) are partially dominant genes; b(7) is partially recessive. Relative strengths of resistance conferred by those genes toward race 1 of the pathogen were B(4) b(7)>B(In) B(2). B(4), B(In), and b(7) each conferred resistance toward X. campestris pv. malvacearum carrying a single avirulence gene, whereas B(2) was less specific.