Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment.

BACKGROUND: Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy. RESULTS: At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C. CONCLUSIONS: Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.

Revealing the importance of meristems and roots for the development of hypersensitive responses and full foliar resistance to Phytophthora infestans in the resistant potato cultivar Sarpo Mira.

The defence responses of potato against Phytophthora infestans were studied using the highly resistant Sarpo Mira cultivar. The effects of plant integrity, meristems, and roots on the hypersensitive response (HR), plant resistance, and the regulation of PR genes were analysed. Sarpo Mira shoots and roots grafted with the susceptible Bintje cultivar as well as non-grafted different parts of Sarpo Mira plants were inoculated with P. infestans. The progress of the infection and the number of HR lesions were monitored, and the regulation of PR genes was compared in detached and attached leaves. Additionally, the antimicrobial activity of plant extracts was assessed. The presented data show that roots are needed to achieve full pathogen resistance, that the removal of meristems in detached leaves inhibits the formation of HR lesions, that PR genes are differentially regulated in detached leaves compared with leaves of whole plants, and that antimicrobial compounds accumulate in leaves and roots of Sarpo Mira plants challenged with P. infestans. While meristems are necessary for the formation of HR lesions, the roots of Sarpo Mira plants participate in the production of defence-associated compounds that increase systemic resistance. Based on the literature and on the presented results, a model is proposed for mechanisms involved in Sarpo Mira resistance that may apply to other resistant potato cultivars.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance.

BACKGROUND: The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). RESULTS: A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. CONCLUSIONS: A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event.