Franklin E. Callahan

Identification of resistance gene analogs in cotton (Gossypium hirsutum L.)

Sequence analyses of numerous plant disease resistance genes have revealed the presence of conserved motifs common to this class of genes, namely a nucleotide binding site (NBS) and leucine rich repeat region. In this study, thirty-three resistance gene analogs (RGAs) were cloned and sequenced from cotton (Gossypium hirsutum L.) following PCR with degenerate primers designed from the conserved NBS motif of plant resistance (R) genes. Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into four distinct classes from which several subgroups were delineated based on nucleic acid sequences. Gene database searches with the consensus protein sequences of each of the four classes and respective subgroups of cotton RGAs revealed their conserved NBS domains and homology to RGAs and known resistance genes from a variety of plant genera. Given the complete lack of knowledge regarding molecular organization of R genes in cotton, the cloned RGAs described here may be useful as probes to map, characterize, and manipulate R genes of the cotton genome.

Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.)

A cytoplasmic mutant of Gossypium barbadense L., cyt-V was characterized at the morphological, cellular, genetic and molecular levels using comparison analysis with v7v7, a nuclear virescent mutant to identify molecular effects of the cyt-V mutation. The yellow phenotype was specific only to leaves in the cyt-V mutant (CM-1-90) but the same phenotype was present in both leaves and cotyledons of v7v7, a nuclear virescent mutant, suggesting that cyt-V and v7v7, had different organ-specific gene actions. Chlorophyll and carotenoid levels of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7v7 true leaves were significantly lower than in the true leaves of PS-7 × CM-1-90, v7v7 × CM-1-90 and PS-7. Anatomical studies of chloroplast showed that CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7v7 lacked grana in the thylakoids of the mesophyll cells. This indicated that chlorophyll and carotenoid levels correlated with chloroplast structure. SDS-PAGE analysis of thylakoid preparations revealed decreases of several granalocalized PSII proteins in CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7v7. cDNA-AFLP differential display studies identified several differentially expressed transcripts in the leaves of reciprocal crosses (PS-7 × CM-1-90, v7v7 × CM-1-90 and CM-1-90 × PS-7 and CM-1-90 × v7v7), including one possessing a high sequence homology to a psbA gene. Western blot analysis further confirmed the absence of D1 protein encoded by psbA in CM-1-90 × PS-7′ CM-1-90 × v7v7 and CM-1-90 true leaves. Overall, we studied cyt-V and v7v7 that both are developmental mutants, as all the virescents of cotton mutants, and as such it was difficult to separate cause and effect in the observation; however, we verified that the source of cyt-V mutation was in chloroplast and elucidated that its gene action was different from v7v7. Results indicated that cyt-V is inherited as a single gene but it affects several chloroplast and nucleus-encoded genes. We identified several transcripts that associated with the cyt-V mutation. This study also suggested that chloroplast-encoded gene products might affect the expression of nuclear genes, possibly at the transcriptional stage.

Molecular cloning, differential expression, and functional characterization of a family of class I ubiquitin-conjugating enzyme (E2) genes in cotton (Gossypium)

Two cDNAs and their corresponding genes (GhUBC1 and GhUBC2) encoding ubiquitin-conjugating enzymes (E2s) have been cloned and characterized from allotetraploid cotton Gossypium hirsutum ((AD)(1) genome). Three additional E2 genes (GaUBC1, GtUBC2, and GrUBC2) have also been identified from diploid cottons Gossypium arboreum (A(2) genome), Gossypium thurberi (D(1) genome), and Gossypium raimondii (D(5) genome), respectively. The derived amino acid sequences of the five closely related cotton E2s are 77-79% identical to yeast ScUBC4 and ScUBC5. The GhUBC1/2 gene family is composed of two members, and genomic origin analysis indicates that GhUBC1 and 2 are individually present in the A and D subgenomes of G. hirsutum. The transcript levels of GhUBC1/2 increased significantly in leaves and flowers at senescence, suggesting that GhUBC1/2 may play a role in the degradation of target proteins that function in the delay of the senescence program. Correlated with high auxin content and auxin-associated effects, GhUBC1/2 are also highly expressed in the youngest leaves, the apical part of lateral roots, and elongating fibers. Genetic complementation experiments revealed that GhUBC1 and 2 can substitute for the function of ScUBC4 and 5 required for the selective degradation of abnormal and short-lived proteins in a yeast ubc4ubc5 double mutant.

Transcript analysis of parasitic females of the sedentary semi-endoparasitic nematode Rotylenchulus reniformis.

Rotylenchulus reniformis, the reniform nematode, is a sedentary semi-endoparasitic nematode capable of infecting >300 plant species, including a large number of crops such as cotton, soybean, and pineapple. In contrast to other economically important plant-parasitic nematodes, molecular genetic data regarding the R. reniformis transcriptome is virtually nonexistant. Herein, we present a survey of R. reniformis ESTs that were sequenced from a sedentary parasitic female cDNA library. Cluster analysis of 2004 high quality ESTs produced 123 contigs and 508 singletons for a total of 631 R. reniformis unigenes. BLASTX analyses revealed that 39% of all unigenes showed similarity to known proteins (E<or=1.0e-04). R. reniformis genes homologous to known parasitism genes were identified and included beta-1,4-endoglucanase, fatty acid- and retinol-binding proteins, and an esophageal gland cell-specific gene from Heterodera glycines. Furthermore, a putative ortholog of an enzyme involved in thiamin biosynthesis, thought to exist solely in prokaryotes, fungi, and plants, was identified. Lastly, 114 R. reniformis unigenes orthologous to RNAi-lethal Caenorhabditis elegans genes were discovered. The work described here offers a glimpse into the transcriptome of a sedentary semi-endoparasitic nematode which (i) provides the transcript sequence data necessary for investigating engineered resistance against R. reniformis and (ii) hints at the existance of a thiamin biosynthesis pathway in an animal.

A novel root-specific gene, MIC-3, with increased expression in nematode-resistant cotton (Gossypium hirsutum L.) after root-knot nematode infection ☆

A full-length cDNA, MIC-3, has been identified from a lambda ZAPII cDNA library constructed from the mRNA of nematode-resistant cotton (Gossypium hirsutum L.) roots after infection with root-knot nematode (Meloidogyne incognita). The putative open reading frame of MIC-3 encoded a protein of 141 amino acids with a calculated molecular mass of 15.3 kDa. Seven alternative polyadenylation sites have been identified for the MIC-3 transcripts, and the major transcripts are the longest ones. The MIC-3 gene contains a single intron within its coding region and belongs to a novel, multi-gene family containing up to six members. Expression of MIC-3 is root localized and specifically enhanced in the nematode induced, immature galls of resistant cotton line M-249, suggesting that MIC-3 may play a critical role in the resistance response to root-knot nematode.

Molecular characterization and temporal expression analyses indicate that the MIC (Meloidogyne Induced Cotton) gene family represents a novel group of root-specific defense-related genes in upland cotton (Gossypium hirsutum L.)

The molecular events underlying the resistance of Upland cotton (Gossypium hirsutum L.) to the root-knot nematode (RKN) are largely unknown. In this report, we further characterize the previously identified MIC3 gene including the identification of 14 related MIC cDNAs in nematode-infected roots of allotetraploid cotton that show >85% identity with MIC3. A time-course analysis of RKN infection in resistant and susceptible cotton lines showed that maximum MIC transcript accumulation occurred immediately prior to the phenotypic manifestation of resistance. MIC expression was not induced by mechanical wounding or by virulent reniform nematode infection. MIC expression was undetectable in cotton leaves undergoing a hypersensitive response to Xanthomonas campestris. A time-course analysis of defense gene expression (PR10, ERF5, CDNS, LOX1, POD4, POD8) in resistant and susceptible cotton roots showed that RKN infection specifically elicits the induction of MIC in resistant roots and not other common defense-signaling pathways. These results suggest that cotton resistance to RKN involves novel defense-signaling pathways and further supports the idea that the MIC genes are intimately involved in this resistance response and represent a group of root-specific defense-related genes in cotton.

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.