Lowell B. Johnson

Prospects for understanding avirulence gene function

Avirulence genes are originally defined by their negative impact on the ability of a pathogen to infect their host plant. Many avirulence genes are now known to represent a subset of virulence factors involved in the mediation of the host-pathogen interaction. Characterization of avirulence genes has revealed that they encode an amazing assortment of proteins and belong to several gene families. Although the biochemical functions of the avirulence gene products are unknown, studies are beginning to reveal the features and interesting relationships between the avirulence and virulence activities of the proteins. Identification of critical virulence factors and elucidation of their functions promises to provide insight into plant defense mechanisms, and new and improved strategies for the control of plant disease.

AvrXa10 Contains an Acidic Transcriptional Activation Domain in the Functionally Conserved C Terminus

The avrXa10 gene of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, is a member of the avrBs3 avirulence gene family and directs the elicitation of resistance in a gene-for-gene manner on rice lines carrying the resistance gene Xa10. The carboxyl (C) terminus of AvrXa10 has a previously undescribed domain that is structurally similar to the acidic activation domain of many eukaryotic transcription factors in addition to three nuclear localization signal (NLS) sequences. Removal of the C-terminal 38 codons containing the putative activation domain, but retaining the NLS sequences, was concomitant with the loss of avirulence activity. The C-terminal coding regions of avrBs3 and avrXa7 can be replaced by the corresponding region of avrXa10, and the genes retained specificity for the resistance genes Bs3 in pepper and Xa7 in rice, respectively. The avrBs3 and avrXa7 avirulence activities of the hybrid genes were also lost upon removal of the terminal 38 codons. When fused to the coding sequence of the Gal4 DNA binding domain, AvrXa10 activated transcription in yeast and Arabidopsis thaliana. Removal of the carboxyl region severely reduced transcriptional activation. AvrXa10 would have to be localized to the host cell nucleus to function autonomously in transcriptional activation. Consistent with this requirement, mutations in all three NLS sequences of avrXa10 caused a loss in avirulence activity. The findings demonstrate the requirement of the C terminus for AvrXa10 function and the potential for the members of this family of avirulence gene products to enter the host nucleus and alter host transcription.

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control

The C Terminus of AvrXa10 Can Be Replaced by the Transcriptional Activation Domain of VP16 from the Herpes Simplex Virus

The avirulence gene avrXa10 of Xanthomonas oryzae pv oryzae directs the elicitation of resistance in a gene-for-gene manner in rice lines carrying the resistance gene Xa10. We have localized a transcriptional activator domain in the C terminus of AvrXa10 by using amino acid replacement mutagenesis. One mutant, with replacements at three hydrophobic amino acid residues in the C-terminal domain, was defective for transcriptional activation in yeast and avirulence activity in rice. The activation domain from the herpes virus protein VP16 restored the ability of the bacteria expressing the hybrid protein to elicit a resistance reaction. Elicitation was specific for Xa10, and the reaction had the hallmarks of the response to AvrXa10. The results indicate that a domain with the properties of a transcriptional activator plays a critical role in AvrXa10 function. The results also indicate that the protein has the potential to interact with the plant transcriptional program, although a role for the domain in the stability or conformation of the protein in the plant cannot be excluded. In a broader sense, the transcriptional activation domain of avrXa10 may represent a prokaryotic version of the acidic transcriptional activation domain, which heretofore has been found exclusively in eukaryotes.

Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants

Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases. A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3′ region was introduced into tobacco plants by Agrobacterium tumefaciens. The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis. Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli. Similar bands were absent in extracts from untransformed control plants. OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny. On a fresh weight basis, the OC-I content was higher in leaves (50 μg/g) than in roots (30 μg/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies. OC-I from both sources was active against papain.

Characterization of a 46 kda insect chitinase from transgenic tobacco

Abstract A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.

Selection of interspecific somatic hybrids of Medicago by using agrobacterium-transformed tissues

Abstract Protoplast fusion was investigated as a means of obtaining somatic hybrids between alfalfa, Medicago sativa L., and two sexually incompatible annual species, M. intertexta L. (Mill.) and M. scutellata L. (Mill.). A selection scheme was developed using genetically stable transgenic fusion partners, each carrying a different antibiotic resistance gene. A kanamycin-resistant transgenic alfalfa plant (RS-K2A5051a) that was phenotypically and karyotypically normal was used as a source of mesophyll protoplasts. Because M. intertexta and M. scutellata do not regenerate to plants from culture, A. rhizogenes was used to produce root cultures of each that were resistant to hygromycin B. The root cultures exhibited chromosome stability and were used for protoplast isolation. Double antibiotic selection for somatic hybrid callus was completed 3–5 weeks after polyethylene glycol fusion. Southern blot analysis with a ribosomal DNA probe confirmed that the selected calli were nuclear hybrids. The regenerability of alfalfa from culture was expressed only in the alfalfa (X) M. intertexta hybrid calli, where embryos and several plantlets were obtained.

Peroxidase activity in healthy and leaf-rust-infected wheat leaves

Abstract Peroxidase activity was determined in primary leaves of healthy and inoculated, near-isogenic wheat lines differing in susceptibility to the leaf rust pathogen, Puccinia recondita (isolate UN1-68B). Inoculated LR10(TC), which developed a low (X) infection type, had peroxidase activity 109% greater than healthy controls by 9 days post-infection. Thatcher, which developed a high (4) infection type, had peroxidase activities only 20–48% greater than healthy controls during the 2–9-day period after infection. Activity, similar in healthy leaves of both lines, increased with leaf age. Total buffer-soluble protein (trichloroacetic acid-precipitable) did not change as the disease developed in either line. Peroxidases from both healthy and inoculated LR10(TC) separated on a gel filtration column into two distinct molecular weight groupos. The ratio of activities in these two groups did not differ between 9-day-rusted LR10(TC) and healthy tissue, although total peroxidase was considerably greater in infected tissues. This suggests that the two forms may be related. The low molecular-weight peroxidase fraction had an average molecular weight of near 35,000 (as determined by gel filtration), while the molecular weight of the other fraction was near 160,000. The physiological significance of the two molecular sizes is unknown.

Heteroplasmy of chloroplast DNA in Medicago

Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones

SummaryAlfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.