Fungal Mitochondrial DNases: Effectors with the Potential to Activate Plant Defenses in Nonhost Resistance

Previous reports on the model nonhost resistance interaction between Fusarium solani f. sp. phaseoli and pea endocarp tissue have described the disease resistance-signaling role of a fungal DNase1-like protein. The response resulted in no further growth beyond spore germination. This F. solani f. sp. phaseoli DNase gene, constructed with a pathogenesis-related (PR) gene promoter, when transferred to tobacco, generated resistance against Pseudomonas syringe pv. tabaci. The current analytical/theoretical article proposes similar roles for the additional nuclear and mitochondrial nucleases, the coding regions for which are identified in newly available fungal genome sequences. The amino acid sequence homologies within functional domains are conserved within a wide array of fungi. The potato pathogen Verticillium dahliae nuclease was divergent from that of the saprophyte, yeast; however, the purified DNase from yeast also elicited nonhost defense responses in pea, including pisatin accumulation, PR gene induction, and resistance against a true pea pathogen. The yeast mitochondrial DNase gene (open reading frame) predictably codes for a signal peptide providing the mechanism for secretion. Mitochondrial DNase genes appear to provide an unlimited source of components for developing transgenic resistance in all transformable plants.

Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy

Sheep with an ARQ/ARQ PRNP genotype at codon positions 136/154/171 are highly susceptible to experimental infection with bovine spongiform encephalopathy (BSE). However, a number of sheep challenged orally or intracerebrally with BSE were clinically asymptomatic and found to survive or were diagnosed as BSE-negative when culled. Sequencing of the full PRNP gene open reading frame of BSE-susceptible and -resistant sheep indicated that, in the majority of Suffolk sheep, resistance was associated with an M112T PRNP variant (TARQ allele). A high proportion (47 of 49; 96 %) of BSE-challenged wild-type (MARQ/MARQ) Suffolk sheep were BSE-infected, whereas none of the 20 sheep with at least one TARQ allele succumbed to BSE. Thirteen TARQ-carrying sheep challenged with BSE are still alive and some have survival periods equivalent to, or greater than, reported incubation periods of BSE in ARR/ARR and VRQ/VRQ sheep.

Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH154RQS, GIRQ211QS, GIRRK222S and GIRRQP240) derived from the wild-type allele (G127I142R154R211Q222S240) by a single-codon mutation, and two (S127IRRQP240 and GM142RRQP240) by a double-codon mutation. A case–control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I142R154R211Q222/IRRQ goats, the odds of scrapie cases in IRQ211Q/IRRQ and IRRK222/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M142RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP240 homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5–18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Prion protein gene polymorphisms in healthy and scrapie-affected Spanish sheep

The Rasa Aragonesa sheep is the second most important Spanish breed after the Merino breed. Reported here is the prion protein (PrP) haplotype frequency distribution for scrapie-related codons (136, 154 and 171) and a sequencing study of the complete PrP gene open reading frame for this breed and six other closely related breeds. The study includes four scrapie-affected sheep flocks belonging to Rasa Aragonesa and Rasa Navarra breeds. Thirty-eight scrapie-affected sheep, 502 healthy sheep from scrapie-affected flocks and 905 sheep from a breed survey were genotyped. The most frequent PrP haplotype in both scrapie and healthy flocks was ARQ, which was found at significantly higher frequency in scrapie-affected sheep. The susceptibility-associated VRQ haplotype was found at low frequencies in six out of eight breeds, but was not present in the 38 scrapie-affected sheep. The resistance-associated ARR haplotype was found in all breeds except one (Ojinegra) at frequencies ⩾14 %. Fourteen amino acid polymorphisms were detected in these Spanish sheep, including the known amino acid substitutions at codons 112, 136, 141, 143, 154, 171 and 176, and new polymorphisms at codons 101 (Q→R), 151 (R→G), 151 (R→H), 172 (Y→D) and 175 (Q→E). Most of the novel polymorphic codons show frequencies lower than 5 %.

First Report of Papaya leaf curl virus Infecting Papaya Plants in Taiwan

Papaya leaf curl disease was first reported in India in 1939 (1). Caused by begomovirus, Papaya leaf curl virus (PaLCV) (2), this disease was discovered in the papaya orchards of southern Taiwan in 2002. Infected papaya developed symptoms such as downward curling of leaves, twisted petioles, vein enation, and stunting. Diseased plants produced small and distorted fruits that tend to fall prematurely. Typical twin virion was observed in the diseased papaya cells by electron microscopy. In addition, our whitefly-transmission test demonstrated that the sweet potato whitefly (Bemisia tabaci) could transmit this virus. For further molecular identification, two opposing primers were selected for the polymerase chain reaction (PCR) detection of PaLCV from the published nucleotide sequences of PaLCV (Genbank Accession No. NC004147) (3). The primer pair, composed of the forward primer 5′ -GCT AGA AAT TAT GTC GAA GCG-3′ and the reverse primer 5′-TCA ACT ACA ACC TGA GGA AAG C-3′, was designed to amplify a PaLCV-specific 1,031-bp fragment containing 774 bp of the coat protein gene open reading frame (CP-ORF) using PCR. Five diseased papaya samples with typical leaf-curl symptoms tested positive in the PCR-based assay with this specific primer pair, whereas five healthy papaya samples tested negative. However, the sequencing results of the PCR product from five PaLCV-infected papayas indicated the CP-ORF of PaLCV in Taiwan (PaLCV-Tw) was somewhat different from PaLCV in India (PaLCV-Id). The DNA sequences (Genbank Accession No. AY183472) of CP-ORF of PaLCV-Tw were 80% identical to those of PaLCV-Id, and their translated amino acid sequences were 77% identical. This indicates that PaLCV-Tw and PaLCV-Id are two different species or strains. References: (1) K. M. Thomas and C. S. Krishnaswamy. Curr. Sci. 8:316,1939. (2) S. Saxena et al. Plant Dis. 82:126, 1998. (3) S. Saxena et al. Biochem. Mol. Biol. Int. 45:101, 1998.

Expression of the Neutral Protease Gene from a Thermophilic Bacillus sp. BT1 Strain in Bacillus subtilis and Its Natural Host: Identification of a Functional Promoter

ABSTRACT The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5′ to the gene. In contrast, in heterologous B. subtilis this thermophilic nprpromoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5′ to ORF X that is required for efficient expression of thenpr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5′ rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis.