Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

<p>Abstract</p> <p>Background</p> <p>Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and <it>in planta </it>experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen <it>Pectobacterium atrosepticum</it>, belonging to the family <it>Enterobacteriaceae</it>.</p> <p>Results</p> <p>Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and <it>in planta</it>. Two of these genes (<it>recA </it>and <it>ffh</it>), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes <it>proC </it>and <it>gyrA</it>, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels.</p> <p>Conclusion</p> <p>Based on these results, we suggest <it>recA </it>and <it>ffh </it>as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen <it>P. atrosepticum </it>and potentially other related pathogens.</p

Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum

The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees

Climate, host and geography shape insect and fungal communities of trees

13 Pág.Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.We gratefully acknowledge the financial support of the Swiss National Science Foundation (Project C15.0081) Grant 174644 and the Swiss Federal Office for the Environment Grant 00.0418.PZ/P193-1077. This work was supported by COST Action “Global Warning” (FP1401). CABI is an international intergovernmental organisation, and R.E., M.K., H.L. and I.F. gratefully acknowledge the core financial support from our member countries (and lead agencies) including the United Kingdom (Foreign, Commonwealth and Development Office), China (Chinese Ministry of Agriculture and Rural Affairs), Australia (Australian Centre for International Agricultural Research), Canada (Agriculture and Agri-Food Canada), Netherlands (Directorate General for International Cooperation), and Switzerland (Swiss Agency for Development and Cooperation). See https://www.cabi.org/aboutcabi/who-we-work-with/key-donors/ for full details. M.B. and M.K.H. were financially supported by the Slovak Research and Development Agency (Project APVV-19-0116). H.B. would like to thank the botanist Jorge Capelo who helped with Myrtaceae identification and INIAV IP for supporting her contribution to this study. Contributions of M. de G. and B.P. were financed through Slovenian Research Agency (P4-0107) and by the Slovenian Ministry of Agriculture, Forestry and Food (Public Forestry Service). G.C, C.B.E. and A.F.M. were supported by OTKA 128008 research grant provided by the National Research, Development and Innovation Office. Contributions of K.A. and R.D. were supported by the Estonian Research Council grants PSG136 and PRG1615. M.J.J., C.L.M. and H.P.R. were financially supported by the 15. Juni Fonden (Grant 2017-N-123). P.B., B.G. and M.Ka. were financially supported by the Ministry of Science and Higher Education of the Republic of Poland for the University of Agriculture in Krakow (SUB/040013-D019). C.N. was financially supported by the Slovak Research and Development Agency (Grant APVV-15-0531). N.K. was partially supported by the Russian Science Foundation (grant № 22-16-00075) [species identification] and the basic project of Sukachev Institute of Forest SB RAS (№ FWES-2021-0011) [data analysis]. R.OH. was supported by funding from DAERA, and assistance from David Craig, AFBI. T.P. thanks the South African Department of Forestry, Fisheries and the Environment (DFFE) for funding noting that this publication does not necessarily represent the views or opinions of DFFE or its employees. In preparing the publication, materials of the bioresource scientific collection of the CSBG SB RAS “Collections of living plants indoors and outdoors” USU_440534 (Novosibirsk, Russia) were used. M.Z. was financially supported by Ministry of Science, Technological Development and Innovation of the Republic of Serbia (contract no. 451-03-47/2023-01/200197). We acknowledge the Genetic Diversity Centre (GDC) at ETH Zurich for providing computational infrastructure and acknowledge the contribution of McGill University and Génome Québec Innovation Center (Montréal, Quebec, Canada) for pair-end sequencing on Illumina MiSeq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Transformation systems, gene silencing and gene editing technologies in oomycetes

Oomycetes are spore-forming eukaryotic microbes responsible for infections in animal and plant species worldwide, posing a threat to natural ecosystems, biodiversity and food security. Genomics and transcriptomics approaches, together with host interaction studies, give promising results towards better understanding of the infection mechanisms in oomycetes and their general biology. Significant development and progress in oomycetes genomic studies have been achieved over the past decades but further understanding of molecular processes, gene regulations and infection mechanisms are still needed. The use of molecular tools such as CRISPR/Cas and RNAi helped elucidate some of the molecular processes involved in host invasion and infection both in plant and animal pathogenic oomycetes. These methods provide an opportunity for accurate and detailed functional analysis involving various fields of studies such as genomics, epigenomics, proteomics, and interactomics. Functional gene characterisation is essential for filling the knowledge gaps in dynamic biological processes. However, every method has both advantages and limitations that should be considered before choosing the best method for investigating a particular research question. Here we review transformation systems, gene silencing and gene editing techniques in oomycetes, how they function, in which species and what are their main advantages and disadvantages

Transformation systems, gene silencing and gene editing technologies in oomycetes

Oomycetes are spore-forming eukaryotic microbes responsible for infections in animal and plant species worldwide, posing a threat to natural ecosystems, biodiversity and food security. Genomics and transcriptomics approaches, together with host interaction studies, give promising results towards better understanding of the infection mechanisms in oomycetes and their general biology. Significant development and progress in oomycetes genomic studies have been achieved over the past decades but further understanding of molecular processes, gene regulations and infection mechanisms are still needed. The use of molecular tools such as CRISPR/Cas and RNAi helped elucidate some of the molecular processes involved in host invasion and infection both in plant and animal pathogenic oomycetes. These methods provide an opportunity for accurate and detailed functional analysis involving various fields of studies such as genomics, epigenomics, proteomics, and interactomics. Functional gene characterisation is essential for filling the knowledge gaps in dynamic biological processes. However, every method has both advantages and limitations that should be considered before choosing the best method for investigating a particular research question. Here we review transformation systems, gene silencing and gene editing techniques in oomycetes, how they function, in which species and what are their main advantages and disadvantages

Transformation systems, gene silencing and gene editing technologies in oomycetes

Oomycetes are spore-forming eukaryotic microbes responsible for infections in animal and plant species worldwide, posing a threat to natural ecosystems, biodiversity and food security. Genomics and transcriptomics approaches, together with host interaction studies, give promising results towards better understanding of the infection mechanisms in oomycetes and their general biology. Significant development and progress in oomycetes genomic studies have been achieved over the past decades but further understanding of molecular processes, gene regulations and infection mechanisms are still needed. The use of molecular tools such as CRISPR/Cas and RNAi helped elucidate some of the molecular processes involved in host invasion and infection both in plant and animal pathogenic oomycetes. These methods provide an opportunity for accurate and detailed functional analysis involving various fields of studies such as genomics, epigenomics, proteomics, and interactomics. Functional gene characterisation is essential for filling the knowledge gaps in dynamic biological processes. However, every method has both advantages and limitations that should be considered before choosing the best method for investigating a particular research question. Here we review transformation systems, gene silencing and gene editing techniques in oomycetes, how they function, in which species and what are their main advantages and disadvantages.publishedVersio