Phloem connectivity and transport are not involved in mature plant resistance (MPR) to Potato Virus Y in different potato cultivars, and MPR does not protect tubers from recombinant strains of the virus.

The aims of this study were: i) to investigate mature plant resistance (MPR) against four strains of Potato virus Y (PVYO, PVYN, PVYNTN and PVYN-Wi) in potato cultivars that differ in maturity (e.g. early or maincrop) at different developmental stages, and ii) to determine whether phloem translocation of photoassimilates at different stages including the source-sink transition influences MPR. The data showed that MPR was functional by the flowering stage in all cultivars, and that the host-pathogen interaction is highly complex, with all three variables (potato cultivar, virus strain and developmental stage of infection) having a significant effect on the outcome. However, virus strain was the most important factor, and MPR was less effective in protecting tubers from recombinant virus strains (PVYNTN and PVYN-Wi). Development of MPR was unrelated to foliar phloem connectivity, which was observed at all developmental stages, but a switch from symplastic to apoplastic phloem unloading early in tuber development may be involved in the prevention of tuber infections with PVYO. Recombinant virus strains were more infectious than parental strains and PVYNTN has a more effective silencing suppressor than PVYO, another factor that may contribute to the efficiency of MPR. The resistance conferred by MPR against PVYO or PVYN may be associated with or enhanced by the presence of the corresponding strain-specific HR resistance gene in the cultivar.

Genotypic and Phenotypic Structure of the Population of Phytophthora infestans in Egypt Revealed the Presence of European Genotypes.

Late blight disease of potato and tomato, caused by Phytophthora infestans, results in serious losses to Egyptian and global potato and tomato production. To understand the structure and dynamics of the Egyptian population of P. infestans, 205 isolates were collected from potato and tomato plants during three growing seasons in 2010-2012. The characterization was achieved by mating-type assay, metalaxyl sensitivity assay, and virulence pattern. Additionally, genotyping of 85 Egyptian isolates and 15 reference UK isolates was performed using 12 highly informative microsatellite (SSR) markers David E. L. Cooke and five effector (RxLR) genes. Mating-type testing showed that 58% (118 of 205) of the isolates belonged to mating type A1, 35% (71 isolates) to mating type A2, and the rest 8% (16 isolates) were self-fertile. The phenotype of metalaxyl response was represented as 45% resistant, 43% sensitive, and 12% as intermediate. Structure analysis grouped the 85 identified genotypes into two main clonal lineages. The first clonal lineage comprised 21 isolates belonging to A2 mating type and 8 self-fertile isolates. This clonal lineage was identified as Blue_13 or EU_13_A2. The second main clonal lineage comprised 55 isolates and was identified as EU_23_A1. A single isolate with a novel SSR genotype that formed a distinct genetic grouping was also identified. The effector sequencing showed good correspondence with the virulence data and highlighted differences in the presence and absence of loci as well as nucleotide polymorphism that affect gene function. This study indicated a changing population of P. infestans in Egypt and discusses the findings in the context of late blight management.

Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.

• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

Excision and episomal replication of cauliflower mosaic virus integrated into a plant genome.

Transgenic Arabidopsis (Arabidopsis thaliana) plants containing a monomeric copy of the cauliflower mosaic virus (CaMV) genome exhibited the generation of infectious, episomally replicating virus. The circular viral genome had been split within the nonessential gene II for integration into the Arabidopsis genome by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were assessed for episomal infections at flowering, seed set, and/or senescence. The infections were confirmed by western blot for the CaMV P6 and P4 proteins, electron microscopy for the presence of icosahedral virions, and through polymerase chain reaction across the recombination junction. By the end of the test period, a majority of the transgenic Arabidopsis plants had developed episomal infections. The episomal form of the virus was infectious to nontransgenic plants, indicating that no essential functions were lost after release from the Arabidopsis chromosome. An analysis of the viral genomes recovered from either transgenic Arabidopsis or nontransgenic turnip (Brassica rapa var rapa) revealed that the viruses contained deletions within gene II, and in some cases, the deletions extended to the beginning of gene III. In addition, many of the progeny viruses contained small regions of nonviral sequence derived from the flanking transformation vector. The nature of the nucleotide sequences at the recombination junctions in the circular progeny virus indicated that most were generated by nonhomologous recombination during the excision event. The release of the CaMV viral genomes from an integrated copy was not dependent upon the application of environmental stresses but occurred with greater frequency with either age or the late stages of plant maturation.

The influence of RNA-dependent RNA polymerase 1 on potato virus Y infection and on other antiviral response genes.

The gene encoding RNA-dependent RNA polymerase 1 (RDR1) is involved in basal resistance to several viruses. Expression of the RDR1 gene also is induced in resistance to Tobacco mosaic virus (TMV) mediated by the N gene in tobacco (Nicotiana tabacum cv. Samsun NN) in an incompatible hypersensitive response, as well as in a compatible response against Potato virus Y (PVY). Reducing the accumulation of NtRDR1 transcripts by RNA inhibition mediated by transgenic expression of a double-stranded RNA hairpin corresponding to part of the RDR1 gene resulted in little or no induction of accumulation of RDR1 transcripts after infection by PVY. Plants with lower accumulation of RDR1 transcripts showed much higher accumulation levels of PVY. Reduced accumulation of NtRDR1 transcripts also resulted in lower or no induced expression of three other antiviral, defense-related genes after infection by PVY. These genes encoded a mitochondrial alternative oxidase, an inhibitor of virus replication (IVR), and a transcription factor, ERF5, all involved in resistance to infection by TMV, as well as RDR6, involved in RNA silencing. The extent of the effect on the induced NtIVR and NtERF5 genes correlated with the extent of suppression of the NtRDR1 gene.

Assessment of CaMV-mediated gene silencing and integration of CaMV into GM plants with a 35S RNA promoter.

Four GM plant species (Arabidopsis thaliana, Brassica napus, Nicotiana benthamiana and N. tabacum), each expressing the gene encoding the jellyfish green fluorescent protein (GFP) regulated by the cauliflower mosaic virus (CaMV) 35S RNA promoter, were assessed for the extent of transgene silencing and viral genome integration following infection by CaMV. The first three species are systemic hosts of CaMV, while N. tabacum is only a local host for a few strains of CaMV. A generalized systemic silencing of the GFP transgene was not observed in a total of 100 plants of each species infected with CaMV, although some localized loss of GFP was observed in CaMV-infected N. benthamiana leaves, and some loss of fluorescence was observed in older leaves of uninfected as well as infected plants. Progeny seedlings obtained from the above infected plants also did not exhibit transgene silencing showing that virus infection did not affect the stability of the transgene. These progeny plants also did not show signs of virus infection, indicating that the presence of the CaMV 35S RNA promoter sequences in the plant genome did not potentiate seed transmission of the virus. Integration of infective CaMV into the CaMV 35S RNA promoter could not be detected in 944 samples taken from leaves of the above infected plant species or in 2912 samples taken from progeny seedlings. Based on a detection limit of one copy per 4000 haploid genomes, we conclude that if integration of virus does occur into the CaMV 35S RNA promoter, then it occurs at such a low frequency as to be insignificant.

Sheep-urine-induced changes in soil microbial community structure.

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.