Task 1 - Histopathological study of Castanea spp.- Pc interactions. Selection of time points for transcriptomics

This task aims to evaluate the progression of Phytophthora cinnamomi ( Pc)infection and the sequence of responses it induced in root tissues of resistant and susceptible Castanea species, respectively C. crenata and C. sativa and 3 hybrids between these two species.

Three months-old plants will be inoculated with Pc following protocols already established. (10). At different times after inoculation, root samples (inoculated and control) will be collected and prepared for light microscopic observations:

  1. Root samples will be previously fixed and rehydrated in distilled water. Pieces and cross sections (25-30 um thick) obtained with a freezing microtome will be submitted to different techniques (blue lactophenol, calcofluor white, Evans blue, phloroglucinol-HCl, epifluorescence and aniline blue tests)(15; 16);
  2. Strips from healthy and infected roots will be submitted to fixation, dehydration, embedding and polymerization in Spurr, Epon or LR White resins. Semi-thin sections (2 um thick) of the polymerised blocks, made using a Pyramitome and an Ultramicrotome will be stained with toluidine blue. (17, 16) Histological data will be used to choose the adequate time points after inoculation for transcriptomic analysis (task 3). Expected results: We expect to unravel the mechanisms that the resistant species C. crenata has, to avoid the progression of Pc and determine the pattern of invasion in the susceptible C.sativa and the hybrids with intermediate phenotypes 

10 - Santos, C, Machado, H, Correia I, Gomes F, Gomes-Laranjo J, and Costa R ( 2015) - Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance. Plant Pathology DOI: 10.1111/ppa.12313.

15 - Jiang RH, Tyler BM (2012) - Mechanisms and evolution of virulence in oomycetes. Annu Rev Phytopathol - 50:295-318

16 - Loureiro A, Nicole M, Várzea V, Moncada P, Bertrand B, Silva MC. (2012) - Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites. Physiological and Molecular Plant Pathology 77: 23-32

17 - Redondo MA, Pérez-Sierra A, Abad-Campos P, Torres L, Solla A, Reig-Armiñana J, García-Breijo F (2015) - Histology of Quercus ilex roots during infection by Phytophthora cinnamomi. Trees. Doi:10.1007/s00468-015-1275-3

18 - Silva MC, Nicole M, Guerra-Guimarães L, Rodrigues Jr. CJ. (2002) - Hypersensitive cell death and post-haustorial defense responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves. Physiological and Molecular Plant Pathology 60 (4): 169-183


Task 2 - Transcript profiling

The objective of this task is to sequence transcriptomes of Castanea RNA seq - inoculated and non-inoculated with P. cinnamomi, to identify differentially expressed genes and to study the pattern of expression in five genotypes of Castanea with a broad-spectrum response to P. cinnamomi: C.crn - resistant, C.sat - susceptible and 3 C.sat x C.crn hybrids with intermediate phenotypes. During the transcriptome construction, unlike the first published data of C. sativa and C. crenata, the root samples will be individually sequenced (avoiding pool RNA), and the different time points and amplitude of the response to pathogen infection, will be chosen based on the histopathological studies (previous task)

Methods: Root transcriptomes will be prepared from biological triplicates of C.sat, C.crn and 3 C.sat x C.sat hybrids, collected at 3 time points after inoculation. The 3 time points after inoculation will comprise different stages of infection, based on the histopathological studies (task 1). Plants with roots under similar physiological conditions will be produced by micropropagation and will be inoculated with a virulent strain of P. cinnamomi, following the protocol already established (10). Total RNA will be isolated from roots using the protocol optimized for the plant material (13). A total of 12 libraries will be sequenced in Illumina platform: 9 inoculated, corresponding to 3 genotypes x 3 timepoints + 3 non inoculated, one for each genotype, all with biological triplicates.

INIAV will be responsible for the production of 36 plants by micropropagation from the 36 genotypes: 27 (3 replicates x 3 genotypes x 3 timepoints) will be inoculated with P. cinnamomi and 9 (3 replicates x 3 genotypes) will not. INIAV will be also responsible for the inoculation assays with P. cinnamomi and RNA extraction and purification. ITQB will be responsible for the sequencing, assembly, annotation and selection of differentially expressed genes. The draft of Castanea mol (Castanea mollissima) genome is available: http://www.hardwoodgenomics.org/organism/Castanea/mollissima

Expected results: We expect to identify differentially expressed genes at different stages of P. cinnamomi infection and analysis of the pattern of differential expression observed for different genotypes, with a broad range of response to the pathogen. Gene expression profiles of the most significant and interesting genes will elucidate the contribution of each gene for the phenotypic differences observed in the 3 genotypes. The transcriptomes will be validated by digital PCR in the next task.

13 - Santos C, Duarte S, Tedesco S, Fevereiro P e Costa L R (2017) - Expression profiling of Castanea genes during resistant and susceptible interactions with the oomycete pathogen Phytophthora cinnamomi reveal possible mechanisms of immunity. Frontiers in Plant Science, doi:10.3389/fpls.2017.00515



Task 3 - Validation of candidate resistance genes by digital PCR

Task 3 - The objective of this task is to validate the most significant differentially expressed genes (DEG) identified in Task 2, in C.crn relatively to C.sat and C.sat x C.crn hybrid, by digitalPCR (dPCR). Digital PCR is a new PCR technique that detects and counts DNA, cDNA or RNA target one molecule at a time. dPCR offers a highly precise method for quantifying copy number variation (absolute quantification), in a very diluted sample without reference to a standard curve. For our purposes, those features constitute great advantages for DEGs validation comparing with qRT-PCR because low yields are obtained from roots in RNA isolation, and reference genes are not identified yet for root Castanea species inoculated with P. cinnamomi. Twenty DEGs will be selected for validation in the 36 plants mentioned in Task 2. The parameters for gene selection will be a fold-change higher than 2 relatively to non-infected plants and the reported relevance to plant resistance, to be considered as candidate genes to pathogen resistance.

Firstly, cDNA will be obtained from each RNA sample isolated following the protocol referred in Task 2, by reverse transcription with oligo(dT) and random primers. Specific primers and probes for each DEG will be designed according digital PCR specifications, using bioinformatics tools. Primers specificity will be evaluated by qRT-PCR using the SYBR green system. dPCR will be based on QuantStudio 3D Digital PCR System userguide. cDNA samples will be diluted and added to dPCR reaction mix. Each amplification reaction will be performed in triplicate. Each dPCR chip is loaded with 14.5 μl of dPCR reaction mixture and then sealed. The dPCR chips are loaded into GeneAmp® PCR System 9700 for 96ºC 10 min, 60ºC 2 min and98ºC 30 sec for 39 cycles, then 60ºC for 2 min and hold at 10ºC. The chips will be read on QuantStudio? 3D DigitalPCR System and data is processed with QuantStudio 3D Analysis Suite, which is available online (https://apps.lifetechnologies.com/quantstudio3d/).

Expected results: We expect to obtain the level of expression of circa 20 candidate genes to pathogen resistance for the 3 time-points upon pathogen inoculation, and consequently the transcript profiling of those genes. If we obtain a higher level of expression for the resistant C.crn relatively to the susceptible C.sat, the result obtained by RNA-seq is validated and the gene is a stronger candidate to pathogen resistance. Gene expression profiles of those genes will elucidate the contribution of each gene for the phenotypic differences observed in the 3 genotypes mentioned and may allow defining a gene network hypothesis for P. cinnamomi resistance. Selection of molecular markers in the validated genes.



Task 4 - Development of a High-Density Genetic Map with GBS markers

The objective of this task is to develop a High-Density Genetic Map for QTL Analysis of Resistance to P. cinnamomi.

Methods: We will make GBS libraries of the population already available of 150 progenies between C. sativa x C crenata. The GBS libraries will be done with biological triplicatesof C.sat and C.crn plus the 150 hybrid progenies obtained from controlled crosses between the two species. Genomic DNA will be isolated in Portugal at INIAV using 2% CTAB protocol and purified using DNeasy Plant Mini kit (QIAGEN). The GBS libraries will be done by LGC company and the Bioinformatics by Marco Ramos at CEBAL.

Expected results: We expect to obtain a huge set of new polymorphic SNPs identified in the C.sat x C.crn segregating population, leading to the construction of high density genetic map which will allow significant QTLs identification for P. cinnamomi. Localization of resistance genes in the genetic linkage map and identification of markers for MAS and also identify the markers linked to resistance for MAS. (to be performed in conjugation with task 3 and 4).



Task 5 - Mass propagation of the new genotypes with improved resistance to Phytophthora cinnamomi

The objective of this task is to produce the plant material necessary for the previous tasks, and also the improved genotypes selected from the breeding program on course, with the new molecular markers developed in task 5.

Methods: The new genotypes, selected with molecular markers, will be validated though the conventional inoculation method with the pathogen (root inoculation of multiple copies of each genotype, produced by micropropagation) using protocols already optimised by the team(10). At this moment we have an optimized pipeline for plant production (19). All the steps of the micropropagation process, namely the multiplication, rooting and acclimatization to ex-vitro conditions were optimized and we are able to produce 3000 plants/year and these numbers may be increased if needed. The new genotypes, molecular assisted selected, will planted in the two field trials installed under the scope of previous projects, located respectively in the North  Trás-os Montes region, at the Municipality of Bragança, the main producing area of chestnut and in the Municipality of Marvão in Alto Alentejo region to evaluate their performance regarding the adaptation to soil and climate conditions of each region, grafting compatibility with regional nut varieties and susceptibility toroot pathogens (to be performed by INIAV).

Expected results: We expect to develop an efficient pipeline for selection, by molecular markers, of improved genotypes of Castanea with improved resistance to P. cinnamomi and with genetic variability for adaptation to different producing regions of Portugal and Europe and compatibility for grafting with different nut varieties. This task will be performed taking profit of the pilot unit for plant production, constitute by a HiTech greenhouse and a demonstrative field, created by project ALT20-03-0246-FEDER-000011 - NEW CastRootstocks, a demonstrative project.

10 - Santos, C, Machado, H, Correia I, Gomes F, Gomes-Laranjo J, and Costa R ( 2015) - Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance. Plant Pathology. DOI: 10.1111/ppa.12313.