Jani Kelloniemi

Why do viruses need phloem for systemic invasion of plants

Plant viruses use sieve elements in phloem as the route of long-distance movement and systemic infection in plants. Plants, in turn, deploy RNA silencing, R-gene mediated defence and other mechanisms to prevent phloem transport of viruses. Cell-to-cell movement of viruses from an initially infected leaf to stem and other parts of the plant could be another possibility for systemic invasion, but it is considered to be too slow. This idea is supported by observations made on viruses that are deficient in phloem loading. The leaf abscission zone forming at the base of the petiole may constitute a barrier that prevents viral cell-to-cell movement. The abscission zone and protective layer are difficult to localize in the petiole until the leaf reaches an advanced stage of senescence. Viruses tagged with the green fluorescent protein are helpful for localization and study of the developing abscission zone.

Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

Inhibition of Angiotensin Converting Enzyme I Caused by Autolysis of Potato Proteins by Enzymatic Activities Confined to Different Parts of the Potato Tuber

Autolysis of protein isolates from vascular bundle and inner tuber tissues of potato (Solanum tuberosum) enhanced the inhibition of the angiotensin converting enzyme I (ACE), a biochemical factor affecting blood pressure (hypertension). The physiological age of the tuber affected the strength of ACE inhibition, the rate of its increase during autolysis, and the tuber tissue where ACE inhibition was most pronounced. The highest inhibitory activities (50% reduction in ACE activity achieved following autolysis at a protein concentration of 0.36 mg mL (-1)) were measured in tubers after 5-6 months of storage prior to sprouting. The rate of ACE inhibition was positively correlated with protease activity in tuber tissues. Amendment of the autolysis reaction with protein substrates from which bioactive ACE-inhibitory peptides may be released, for example, a purified recombinant protein or a concentrate of total tuber proteins, also enhanced ACE inhibition. Many tuber proteins including aspartic protease inhibitors were degraded during autolysis. The data provide indications of differences in the enzymatic activities confined to different parts of the potato tuber at different physiological stages. Results suggest that native enzymes and substrate proteins of potato tubers can be utilized in search of dietary tools to manage elevated blood pressure.

First Report of Streptomyces europaeiscabiei Causing Common Scab on Potato in Finland

Common scab is an important disease in potato (Solanum tuberosum) caused by Streptomyces spp. In Finland, morphological and physiological characterization (5) and comparison of the 16S rRNA gene sequences have suggested that Streptomyces scabies and S. turgidiscabies are the main causal species (2), but occurrence of S. europaeiscabiei has not been previously studied. In September 2011, potato tubers of cvs. Fambo, Melody, Puikula, Rosamunda, Victoria, and Van Gogh showing symptoms of common scab were collected from 10 fields in southern (Lammi), eastern (Liperi), and western Finland (Isokyrö, Kalajoki, Tyrnävä). Symptoms included superficial, raised, and pitted corky lesions ranging from 3 to 10 mm in diameter. Isolations were made from scab lesions on water agar. Colonies phenotypically characteristic of Streptomyces were transferred to glucose yeast malt extract agar (GYM) after 7 days. Pure cultures were obtained through subsequent transfers to fresh GYM medium, on which the strains produced golden brown colonies and white to grey spores. DNA was extracted from bacteria using the E.Z.N.A. SP Plant DNA Mini Kit (Omega Bio-Tek, Inc.). Primers developed for the 16S rRNA gene sequences (4) were used to detect S. scabies and S. turgidiscabies by PCR. Of the 14 strains recovered, nine were assigned to S. scabies, three to S. turgidiscabies, and two remained unidentified. However, S. scabies and S. europaeiscabiei cannot be distinguished by the 16S rRNA gene sequence, but the ITS1 region of the 16S operon sequence amplified by PCR is cleaved by Hpy99I in S. scabies but not in S. europaeiscabiei (1). Altogether, 18 strains were tested, including nine obtained in 2011 and seven Finnish and two Swedish strains isolated and assigned to S. scabies in the mid-1990s (2,4,5). The ITS1 sequence of S. scabies type strain, ATCC49173, was cleaved with Hpy99I, in contrast to all other strains that were consequently assigned to S. europaeiscabiei. To further confirm the identity of the Finnish strains, sequences of atpD, recA, and rpoB genes from three strains (one from 1995, two from 2011) (GenBank Accession Nos. KJ802471 to 79) were found to be 100, 99.8, and 100% identical, respectively, to corresponding S. europaeiscabiei type strain CFBP 4497 sequences (3). Pathogenicity of the S. europaeiscabiei strains isolated in 2011 was confirmed using radish seedling assay (1). All strains prevented or reduced the growth of radish seedlings (cv. French Breakfast) or caused severe necrosis in repeated experiments. No symptoms were observed on the seedlings grown on oat meal agar without bacteria. The pathogenicity of the S. europaeiscabiei strains isolated in the mid-1990s was confirmed using minituber assays (4,5). In addition, two of these strains were tested in a glasshouse experiment and two in a radish seedling assay and shown to be pathogenic. The results suggest that S. europaeiscabiei is an important cause of potato common scab in Finland. In the earlier studies, S. europaeiscabiei may have been mistaken for S. scabies, as the restriction analysis of the ITS1 region was not done. References: (1) R. Flores-Gonzáles et al. Plant Pathol. 57:162, 2008. (2) J. F. Kreuze et al. Phytopathology 89:462, 1999. (3) D. P. Labeda. Int. J. Syst. Evol. Microbiol. 61:2525, 2011. (4) M. J. Lehtonen et al. Plant Pathol. 53:280, 2004. (5) P. Lindholm et al. Plant Dis. 81:1317, 1997.

Repeated Applications of a Nonpathogenic Streptomyces Strain Enhance Development of Suppressiveness to Potato Common Scab

Potato common scab caused by several Streptomyces spp. is an important disease with no effective methods of control. Suppressiveness against common scab can develop in soil as a result of long-term potato monoculture and has been associated with nonpathogenic Streptomyces spp. To determine whether the development of scab suppressiveness could be enhanced, the effect of repeated applications of an antagonistic Streptomyces strain on common scab was investigated in a long-term field trial over 5 years. Streptomyces strain 272 applied annually at planting consistently suppressed development of common scab symptoms. On scab-susceptible potato cultivar Bintje, strain 272 reduced disease severity, on average, by 43%; whereas, on the scab-tolerant Nicola, the strain reduced both disease incidence and severity by 43 and 59%, respectively. Regardless of disease pressure, the combined use of strain 272 and the tolerant cultivar reduced the scab coverage to a negligible level. After a single application of strain 272, efficient disease suppression did not persist in the soil to the following growing season. However, when strain 272 was applied in three or more consecutive years, the soil remained suppressive to scab for at least 2 years beyond the last application, suggesting that, with repeated applications, it may be possible to enhance development of scab suppression in soil.