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Normalization of data, by choosing the appropriate reference genes (RGs), is fundamental for obtaining reliable results in reverse transcription-quantitative PCR (RT-qPCR). In this study, we assessed Actinidia deliciosa leaves inoculated with two doses of Pseudomonas syringae pv. actinidiae during a period of 13 days for the expression profile of nine candidate RGs. Their expression stability was calculated using four algorithms: geNorm, NormFinder, BestKeeper and the deltaCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A (PP2A) were the most stable genes, while β-tubulin and 7s-globulin were the less stable. Expression analysis of three target genes, chosen for RGs validation, encoding the reactive oxygen species scavenging enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) indicated that a combination of stable RGs, such as GAPDH and PP2A, can lead to an accurate quantification of the expression levels of such target genes. The APX level varied during the experiment time course and according to the inoculum doses, whereas both SOD and CAT resulted down-regulated during the first four days, and up-regulated afterwards, irrespective of inoculum dose. These results can be useful for better elucidating the molecular interaction in the A. deliciosa/P. s. pv. actinidiae pathosystem and for RGs selection in bacteria-plant pathosystems.

For plant pathogenic bacteria, adaptation to the apoplast is considered as key in the establishment of the parasitic lifestyle. Pseudomonas syringae pv. actinidiae (Psa), the causal agent of the bacterial canker of kiwifruit, uses leaves as the entry site to colonize plants. Through a combined approach based on 2-DE, nanoLC-ESI-LIT-MS/MS and quantitative PCR, we investigated Psa colonization of the Actinidia deliciosa "Hayward" leaf apoplast during the bacterial biotrophic phase. A total of 58 differentially represented protein species were identified in artificially inoculated leaves. Although the pathogen increased its population density during the initial period of apoplast colonization, plant defense mechanisms were able to impede further disease development. We identified a concerted action of different proteins mainly belonging to the plant defense and metabolism category, which intervened at different times and participated in reducing the pathogen population. On the other hand, bacterial BamA was highly represented during the first week of leaf apoplast colonization, whereas OmpA and Cpn60 were induced later. In addition to presenting further proteomic information on the molecular factors actively participating in this pathosystem, our data characterize the early events of host colonization and will promote the eventual development of novel bioassays for pathogen detection in kiwiplants., Biological Significance: This original study evaluates on a proteomic perspective the interaction occurring into the leaf apoplast between Actinidia deliciosa and its specific pathogen Pseudomonas syringae pv. actinidiae. Despite the initial bacterial multiplication, a concerted action of the plant defense mechanisms blocked the infection during 21days of apoplast colonization, as revealed by the number of differentially-represented proteins identified in artificially-inoculated and control leaves. Three bacterial proteins were also recognized among the over-represented molecules in infected plants. This study may contribute to improve breeding programs aimed at selecting resistant/tolerant kiwifruit cultivars toward P. syringae pv. actinidiae, which present a high representation of the plant proteins here shown to be involved in resistance mechanisms. In addition to present additional information on the molecular players actively participating in this pathosystem, our data will also facilitate the technological development of future bioassays for the detection of this pathogen in kiwiplants., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A pandemic, very aggressive population of Pseudomonas syringae pv. actinidiae is currently causing severe economic losses to kiwifruit crops worldwide. Upon leaf attack, this Gram-negative bacterium systemically reaches the plant shoot in a week period. In this study, combined 2-DE and nanoLC-ESI-LIT-MS/MS procedures were used to describe major proteomic changes in Actinidia chinensis shoot following bacterial inoculation in host leaf. A total of 117 differentially represented protein spots were identified in infected and control shoots. Protein species associated with plant defence, including type-members of the plant basal defence, pathogenesis, oxidative stress and heat shock, or with transport and signalling events, were the most represented category of induced components. Proteins involved in carbohydrate metabolism and photosynthesis were also augmented upon infection. In parallel, a bacterial outer membrane polypeptide component was identified in shoot tissues, whose homologues were already linked to bacterial virulence in other eukaryotes. Semiquantitative RT-PCR analysis confirmed expression data for all selected plant gene products. All these data suggest a general reprogramming of shoot metabolism following pathogen systemic infection, highlighting organ-specific differences within the context of a general similarity with respect to other pathosystems. In addition to present preliminary information on the molecular mechanisms regulating this specific plant-microbe interaction, our results will foster future proteomic studies aimed at characterizing the very early events of host colonization, thus promoting the development of novel bioassays for pathogen detection in kiwifruit material., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A recent re-emerging bacterial canker disease incited by Pseudomonas syringae pv. actinidiae (Psa) is causing severe economic losses to Actinidia chinensis and A. deliciosa cultivations in southern Europe, New Zealand, Chile and South Korea. Little is known about the genetic features of this pathovar. We generated genome-wide Illumina sequence data from two Psa strains causing outbreaks of bacterial canker on the A. deliciosa cv. Hayward in Japan (J-Psa, type-strain of the pathovar) and in Italy (I-Psa) in 1984 and 1992, respectively as well as from a Psa strain (I2-Psa) isolated at the beginning of the recent epidemic on A. chinensis cv. Hort16A in Italy. All strains were isolated from typical leaf spot symptoms. The phylogenetic relationships revealed that Psa is more closely related to P. s. pv. theae than to P. avellanae within genomospecies 8. Comparative genomic analyses revealed both relevant intrapathovar variations and putative pathovar-specific genomic regions in Psa. The genomic sequences of J-Psa and I-Psa were very similar. Conversely, the I2-Psa genome encodes four additional effector protein genes, lacks a 50 kb plasmid and the phaseolotoxin gene cluster, argK-tox but has acquired a 160 kb plasmid and putative prophage sequences. Several lines of evidence from the analysis of the genome sequences support the hypothesis that this strain did not evolve from the Psa population that caused the epidemics in 1984-1992 in Japan and Italy but rather is the product of a recent independent evolution of the pathovar actinidiae for infecting Actinidia spp. All Psa strains share the genetic potential for copper resistance, antibiotic detoxification, high affinity iron acquisition and detoxification of nitric oxide of plant origin. Similar to other sequenced phytopathogenic pseudomonads associated with woody plant species, the Psa strains isolated from leaves also display a set of genes involved in the catabolism of plant-derived aromatic compounds.