Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato.

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5' and 3' ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Mobilization of horizontally acquired island 2 is induced in planta in the phytopathogen Pectobacterium atrosepticum†SCRI1043 and involves the putative relaxase ECA0613 and quorum sensing.

Integrative and conjugative elements (ICEs) contribute to the rapid evolution of bacterial pathogens via horizontal gene transfer of virulence determinants. ICEs have common mechanisms for transmission, yet the cues triggering this process under natural environmental or physiological conditions are largely unknown. In this study, mobilization of the putative ICE horizontally acquired island 2 (HAI2), present in the chromosome of the phytopathogen Pectobacterium atrosepticum SCRI1043, was examined during infection of the host plant potato. Under these conditions, mobilization of HAI2 increased markedly compared with in vitro cultures. In planta-induced mobilization of HAI2 was regulated by quorum sensing and involved the putative ICE-encoded relaxase ECA0613. Disruption of ECA0613 also reduced transcription of genes involved in production of coronafacic acid (Cfa), the major virulence factor harboured on HAI2, whereas their expression was unaffected in the quorum-sensing (expI) mutant. Thus, suppression of cfa gene expression was not regulated by the mobilization of the ICE per se, but was due directly to inactivation of the relaxase. The identification of genetic factors associated solely with in planta mobilization of an ICE demonstrates that this process is highly adapted to the natural environment of the bacterial host and can influence the expression of virulence determinants.

Molecular characterisation of an endornavirus from Rhizoctonia solani AG-3PT infecting potato.

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a soil-borne plant pathogenic fungus that has a broad host range, including potato. In this study, the double-stranded RNA (dsRNA) profiles were defined for 39 Rhizoctonia solani isolates representative of two different anastomosis groups (AGs) associated with black scurf of potato in New Zealand. A large dsRNA of c. 12 kb-18 kb was detected in each of the isolates, regardless of AG or virulence on potato. Characterisation of the large dsRNA from R. solani AG-3PT isolate RS002, using random amplification of total dsRNA and analyses of overlapping cDNA sequences, resulted in the assembly of a consensus sequence of 14 694 nt. A single, large open reading frame was identified on the positive strand of the assembled sequence encoding a putative polypeptide of at least 4893 amino acids, with a predicted molecular mass of 555.6 kDa. Conserved domains within this polypeptide included those for a viral methyltransferase, a viral RNA helicase 1 and an RNA-dependent RNA polymerase. The domains and their sequential organisation revealed the polyprotein was very similar to those encoded by dsRNA viruses of the genus Endornavirus, in the family Endornaviridae. This is the first report of an endornavirus in R. solani, and thus the putative virus is herein named Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial characterisation of the large dsRNAs in five additional AG-3PT isolates of R. solani also identified them as probable endornaviruses, suggesting this family of viruses is widespread in R. solani infecting potato. The ubiquitous nature of endornaviruses in this plant pathogen implies they may have an important, but yet uncharacterised, role in R. solani.

GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

Inactivation of PbTopo IIIß causes hyper-excision of the Pathogenicity Island HAI2 resulting in reduced virulence of Pectobacterium atrosepticum.

Topoisomerase III enzymes are present only in a limited set of bacteria and their physiological role remains unclear. Here, we show that PbTopo IIIß, a homologue of topoisomerase III encoded on the chromosome of Pectobacterium atrosepticum strain SCRI1043 (Pba SCRI1043), is involved in excision of HAI2, a discrete ~100 kb region, from the Pba SCRI1043 chromosome. HAI2 is a Pathogenicity Island (PAI) that encodes coronafacic acid (Cfa), a major virulence determinant required for infection of potato. PAIs are horizontally acquired genetic elements that in some instances are able to excise from the chromosome of their host cell to form a circular episome prior to transfer to a recipient bacterium. We demonstrate excision of HAI2 from the chromosome, a process that is independent of growth phase and that results in the production of a circular intermediate. Inactivation of PbTopo IIIß causes a 10(3) - to 10(4) -fold increase in excision, leading to reduced fitness in vitro and a decrease in the virulence of Pba SCRI1043 on potato. These results suggest that PbTopo IIIß is required for stable maintenance of HAI2 in the chromosome of Pba SCRI1043 and may control as yet unidentified genes involved in viability and virulence of Pba SCRI1043 on potato.