Abstract

   The pathological interaction between oak trees and Phytophthora
   cinnamomi has implications in the cork oak decline observed over the
   last decades in the Iberian Peninsula. During host colonization, the
   phytopathogen secretes effector molecules like elicitins to increase
   disease effectiveness. The objective of this study was to unravel the
   proteome changes associated with the cork oak immune response triggered
   by P. cinnamomi inoculation in a long-term assay, through SWATH-MS
   quantitative proteomics performed in the oak leaves. Using the
   Arabidopis proteome database as a reference, 424 proteins were
   confidently quantified in cork oak leaves, of which 80 proteins showed
   a p-value below 0.05 or a fold-change greater than 2 or less than 0.5
   in their levels between inoculated and control samples being considered
   as altered. The inoculation of cork oak roots with P. cinnamomi
   increased the levels of proteins associated with protein-DNA complex
   assembly, lipid oxidation, response to endoplasmic reticulum stress,
   and pyridine-containing compound metabolic process in the leaves. In
   opposition, several proteins associated with cellular metabolic
   compound salvage and monosaccharide catabolic process had significantly
   decreased abundances. The most significant abundance variations were
   observed for the Ribulose 1,5-Bisphosphate Carboxylase small subunit
   (RBCS1A), Heat Shock protein 90–1 (Hsp90-1), Lipoxygenase 2 (LOX2) and
   Histone superfamily protein H3.3 (A8MRLO/At4G40030) revealing a
   pertinent role for these proteins in the host-pathogen interaction
   mechanism. This work represents the first SWATH-MS analysis performed
   in cork oak plants inoculated with P. cinnamomi and highlights host
   proteins that have a relevant action in the homeostatic states that
   emerge from the interaction between the oomycete and the host in the
   long term and in a distal organ.

Introduction

   The soil-borne oomycete Phytophthora cinnamomi infects the roots of
   cork oak (Quercus suber) plants, induces necrotic lesions, and the loss
   of fine roots [[34]1,[35]2]. This evidence, combined with other
   factors, are the hallmark for the decline of the cork oak savanna-like
   ecosystem in Portugal (cork oak montado) and Spain (cork oak dehesa).
   Climate changes is reducing water availability (drought) [[36]3], and
   the effectiveness of roots in absorbing water is affected by the health
   status of the plant [[37]4,[38]5], which can become less effective in
   accessing groundwater during drought [[39]6]. Insect colonization
   [[40]7] and fungal infections [[41]8,[42]9] can weaken the tree's
   defence system and thus contribute to the decline. To help maintain the
   sustainability of the cork oak agro-forests, the recommended focus is
   to adopt good management practices [[43]10].

   During inter and intracellular cork oak colonization by P. cinnamomi,
   small 10 kDa proteins (elicitins) are secreted by the oomycete and
   increases disease effectiveness. This has been demonstrated by studying
   a β-cinnamomin silenced P. cinnamomi strain, which acted as a weaker
   pathogen against cork oak when compared to the virulence revealed by
   the wild type [[44]11,[45]12]. In the roots of the narrow-leafed lupin
   (Lupinus angustifolius) infected with P. cinnamomi, the expression of
   β-cinnamomin starts to be detected as early as 24 h post-inoculation
   and follows the development of the mycelium into the host, anchored to
   a mycelial cell wall protein, emphasizing the recognition of these
   proteins as virulence factors [[46]13]. However, effector molecules
   from the RxLR, CRN (for Crinkling and Necrosis) and Nep1-like (NLPs)
   protein families are also potentially secreted, encoded by the 171
   RxLR, 72 NLPs and 29 CRN putative genes present in the genomes (78 Mb)
   of three P. cinnamomi isolates, being able to suppress or bypass the
   plant basic defence responses [[47]14]. The molecular mechanisms by
   which the effector molecules act are largely unknown, although the
   entry of some effector proteins into the plant host cells is known to
   follow a mechanism of endocytosis after binding to receptor molecules
   of phosphatidylinositol-3-phosphate (PI-3-P) mediated by the effector
   RxLR domain [[48]15,[49]16]. In the nucleus, the effectors control
   reactions that trigger host cell death or hypersensitive responses (HR)
   [[50]17,[51]18], and in the nucleolus, they can act as modulators of
   histone acetyltransferases (HAT) to reprogram the plant defence gene
   expression and promote infection [[52]19].

   Following compatible or incompatible reactions with plants, oomycete
   compounds like lipids or carbohydrates referred to as
   Pathogen-Associated Molecular Patterns (PAMPs) and effector
   biomolecules elicit local resistance responses or
   PAMPs/effector-triggered immunity (PTI/ETI) in their hosts [[53]20]. In
   Q. suber root cells, during the first 24 h of interaction with P.
   cinnamomi, metabolic patterns undergo a non-linear variation for
   compounds with carbohydrate, glycoconjugate and lipid groups [[54]21].
   At the transcriptomic level, the differential expression of genes
   encoding pathogenesis-related proteins was observed in avocado roots
   challenged with P. cinnamomi [[55]22] and in stem tissues of Eucalyptus
   nitens infected with P. cinnamomi [[56]23]. In a more detailed analysis
   of the transcriptome of chestnut roots inoculated with P. cinnamomi,
   the multiplicity of the defence responses becomes evident with the
   identification of genes related to the HR (hypersensitive response),
   cell wall strengthening, synthesis of flavonoids and systemic acquired
   resistance [[57]24]. Further, resistance (R) genes coding to
   transmembrane proteins such as LRR receptor-like
   serine/threonine-protein kinase in two Castanea species [[58]24] and
   CC-NB-LRR (coiled coil-nucleotide binding-leucine rich repeat) in cork
   oak [[59]25] are also potentially associated to the recognition of
   effector molecules, eventually interacting, according to the
   gene-for-gene model [[60]26]. Activation of these resistance proteins
   can result in the activation of mitogen-activated protein kinase (MAPK)
   signal transduction cascades, leading to transcription factor
   activation and transcription of responsive genes, and these cascades
   can also be activated by proteins sensitive to the production of
   reactive oxygen species (ROS, O[2]^-, H[2]O[2]) [reviewed by
   [61]20,[62]27–[63]29].

   Salicylic acid (SA)/salicylate is also a signaling molecule that plays
   a central role in PAMPs/effector-triggered immunity (PTI/ETI) and in
   the systemic acquired resistance (SAR). SAR is a type of immunity that
   extends to the entire plant beyond the site of infection, protecting
   the plant against a broad spectrum of pathogens [[64]30,[65]31]. The
   expression of a large number of pathogenesis-related genes is activated
   by nuclear transcription factors interacting with NPR1 monomers
   (nonexpressor of pathogenesis related 1), known as the main regulatory
   molecule of the SA-signaling pathway [[66]32,[67]33].

   To overcome the harmful implications of P. cinnamomi on susceptible
   species of thousands of plants worldwide, one of the current challenges
   is the identification of molecular markers or physiological processes
   suitable for recognition of resistant or susceptible host plant species
   or varieties. Information about the constitutive expression level of
   pathogenesis-related genes in non-infected hosts and the reaction time
   mediating the recognition of the invader and the activation of local
   and systemic defence systems can contribute to this global goal, and
   was critical for the recognition of Castanea crenata as a less
   susceptible species than C. sativa [[68]28]. In less susceptible
   avocado rootstocks, the physical and chemical composition of the host's
   tissues at the site of infection was critical to the effectiveness of
   P. cinnamomi zoospore germination and penetration, as the early
   deposition of callose instead of lignin near the site of hyphae
   penetration along the cell wall hindered the development of the
   oomycete's hyphae [[69]34].

   The hypothesis of the present study is that after inoculation of plant
   roots with a pathogen, an immune response is initiated that will lead
   to a new homeostatic state, with protein changes that can be detectable
   in the long-term, distally from the infection site. The aim was to
   identify and quantify proteins in the leaves of cork oak plants
   inoculated with P. cinnamomi in the roots and compare them to those in
   the leaves of non-inoculated plants, at 248 days post-inoculation,
   using SWATH-MS proteomics [[70]35]. SWATH-MS (Sequential Window
   Acquisition of all Theoretical Mass Spectra) is a quantitative,
   label-free and unbiased proteomics method that is able to acquire
   information about virtually every ion (in this case peptides),
   introduced into the mass spectrometer [[71]36]. SWATH is a promising
   strategy for the quantitative screening of a large number of proteins
   that has previously been applied in the field of plant biology
   [[72]37–[73]39] and recognized as a valuable tool for the comprehensive
   study of proteins in plants [[74]40,[75]41].

   The leaves are a distal organ that can be sampled in a minimally
   invasive way in adult trees, so they can also be a potential organ for
   practical monitoring of infection or resistance. Four hundred and
   twenty-four proteins were identified in the cork oak leaves, and a
   subset of 80 proteins showed differential levels between inoculated and
   control plants, being considered responsive to P. cinnamomi. These
   included 18 proteins associated with several gene ontologies (GO)
   biological processes, and their potential role in the cork oak immune
   response is discussed. The GO cellular component “stromules” was also
   significantly enriched among the differential proteins, indicating that
   communication between cellular organelles may be important in the cork
   oak immune response to P. cinnamomi.

Materials and methods

   The design of the project included several experimental procedures
   operated at different time points. In the first phase, the biological
   material was prepared consisting of twelve cork oak seedlings,
   germinated from seeds, with half of these plants being inoculated with
   P. cinnamomi. The following phases started 248 days after inoculation
   and included the harvesting of the leaves from each plant for protein
   extraction and subsequent SWATH-MS proteomics. The experimental assay
   ended with the bioinformatic annotation and quantification of proteins
   present in the extracts of each plant.

Biological material

   Cork oak plants used in this experimental project were germinated from
   acorns taken from six cork oak trees located in Cachopo, Algarve,
   Portugal ([76]S1 Fig). Parental cork oak trees referenced as S1.1,
   S2.1, S4.1, S5.1, S7.1, and S8.1 showed signs of decline at distinct
   stages of progression, based on visual observation of the canopy
   defoliation level typical of P. cinnamomi infection. The study included
   two experimental conditions with six biological replicates: 6 cork oak
   plants inoculated with the PA45 P. cinnamomi isolate and 6
   non-inoculated plants. Seeds from six parental cork oak trees were
   germinated and were distributed between the control and inoculated
   groups so that each inoculated plant had a paired control from the same
   progenitor. [77]S1 Table provides the cork oak references used in the