4 DISCUSSION
The goal of our research was to utilize QTL analysis as a tool to
identify regions of the Populus genome that were important in
mediating biotic interactions. Upon identification of these regions we
were able to directly compare the parental genomes of the hybrid cross
to look for similarity in content and potential gene-for-gene
interactions reflected in recent tandem duplication expansion. We found
that the host plant genotype had a significant effect on fungi and
insects in our study. The progeny segregated for varying resistance to
fungal and insect pathogens and pests that were inherited from the
grandparents and parents of the 52-124 family cross. Novel alleles (i.e.
those from the grandparent that was not native to the region where the
trials occurred) were important for resistance to Melampsora sp.
fungus, M. vagabunda galls, and Phyllocolpa sp.
oviposition. In contrast, the interval associated with S. musivacanker symptoms was the only case in which susceptibility to the fungus
was dominant and inherited from the non-coevolved host (P.
trichocarpa ), as has been previously observed (Muchero et al., 2018;
George Newcombe & Ostry, 2001a).
We detected a major QTL for Melampsora sp. resistance on Chr04.
This region is known to contain the MXC3 locus which confers
resistance to infection of many species of the Melampsora leaf
rusts (George Newcombe, Stirling, & Bradshaw, 2001; Yin et al., 2004).
Based on mean parental infection scores and the allelic effects at the
QTL, progeny in family 52-124 inherited this resistance from theP. trichocarpa grandmother 93-968. Furthermore, the QTL interval
contained two tandem repeats of stigma-specific proteins (Stig1) in both
the P. trichocarpa genome and the orthologous interval in theP. deltoides genome. Several of these showed evidence of positive
selection based on Ka/Ks ratios (Table 5). Stigma-specific proteins,
specifically Stig1, have been found to be associated with female
sterility in tobacco (Nicotiana tabacum ) and petunia
(Petunia hybrida ) (Goldman, Goldberg, & Mariani, 1994; Verhoeven
et al., 2005). Stig1 is known to mediate secretion of exudate lipids in
the intercellular spaces and high expression of the protein inhibits
pollen grains from penetrating style tissue preventing fertilization
(Verhoeven et al., 2005). Most lipid transfer proteins, such as Stig1,
are important in plant cell-wall loosening and their expression can
prevent penetration of plant tissues (Nieuwland et al., 2005).
Diversification of the protein family containing Stig1 may play an
important role in lowering fungal infection in Populus by
providing a physical barrier to resist the Melampsora sp. hyphae.
Another protein domain that was found to be enriched in the P.
trichocarpa Chr04 interval was the malonyl-CoA decarboxylase C-terminal
domain. Similarly, the gene ontology function for malonyl-CoA
decarboxylase activity was also enriched in P. trichocarpa in the
same interval. Genes which are capable of transforming malonyl-CoA could
be beneficial in resistance to Melampsora sp. as it is an
important precursor in the production of several pathogen defensive
compounds, such as isoprenoids in the Mevalonate pathway (H. Chen, Kim,
Weng, & Browse, 2011; Dixon, 2001).
Interestingly, an overlapping interval on Chr04 was found to be
associated with the activity of the leaf spot symptoms of the S.
musiva fungus. Upon further investigation we found that trees that were
not infected by the S. musiva leaf spot had more severe symptoms
of the Melampsora sp. leaf rust. This suggests that a competitive
interaction occurred between the two pathogens in the field during the
year of survey, and was reflected in a false association on Chr04 with
the S. musiva leaf spot score. Competition among fungal pathogens
is not uncommon in field conditions with most examples focused on
different genotypes within the same fungal species (Abdullah et al.,
2017). The outcome of within host tissue colonization of multiple
strains or species often relies upon the genetic similarity of the
pathogens (Abdullah et al., 2017; Koskella, Giraud, & Hood, 2006). In
our case, we had two very different pathogens that were utilizing the
leaf tissue in vastly dissimilar ways. S. musiva is a
necrotrophic fungus, which requires dead tissue to reproduce, andMelampsora sp. is biotrophic, requiring living tissue to
reproduce. The presence of S. musiva on the trees had a much
larger effect on the occurrence of Melampsora sp. infection. This
may indicate that the S. musiva fungus had a competitive
advantage over Melampsora sp. in utilization of thePopulus leaf tissue at our site. Further supporting this finding
was the complete loss of Melampsora sp. symptoms at the
plantation site over the course of ten years and the continual presence
of the S. musiva leaf spot. A similar interaction has been
reported in wheat between the biotroph Blumeria
graminis f.sp. tritici (Bgt ), the powdery mildew
pathogen, and necrotroph Zymoseptoria tritici , the cause ofSeptoria tritici blotch (Orton & Brown, 2016). Similarly, the
outcome of the interaction of the two pathogens in wheat was
competitive. However, the necrotroph was found to actually be capable of
reducing the reproductive capability of the biotroph which indicates
pathogen-pathogen interactions can be more direct rather than relying
solely on the host plant genetics (Orton & Brown, 2016).
The S. musiva stem canker symptoms were found to be associated
with a QTL interval on Chr16. In this study, susceptibility to the
necrotroph appeared to be originating from the presence of P.
trichocarpa alleles in the progeny. Previous work on a similarPopulus hybrid cross supports this with susceptibility toS. musiva stem canker originating primarily from dominant alleles
derived from P. trichocarpa (G. Newcombe, 1998; George Newcombe
& Ostry, 2001a). Interestingly, the loci conferring susceptibility in
family 52-124 did not overlap with the four loci that were uncovered in
a previous genome-wide association study of S. musivasusceptibility in P. trichocarpa (Muchero et al., 2018),
suggesting that different mechanisms may be involved in hybrid
interactions with this pathogen. However, the QTL did contain a tandem
repeat of a G-type lectin receptor-like protein kinase that was expanded
in P. deltoides . This protein could play a similar role to a
receptor-like kinase from the same family (Yang et al., 2016) that was
associated with susceptibility to S. musiva in the P.
trichocarpa study (Muchero et al., 2018).
M. vagabunda has been recorded completing its life cycle on
several species of Populus including P. deltoides andP. tremuloides (Floate, 2010). Although the aphid’s life history
has been documented, little is known about the influence of host plant
genetics on gall formation or resistance to feeding (Floate, 2010;
Ignoffo & Granovsky, 1961). We detected a QTL on Chr05 in which alleles
inherited from P. deltoides were positively associated with gall
occurrence. Genes conferring lipoxygenase and oxidoreductase activity
were enriched in both the P. deltoides as well as the P.
trichocarpa QTL intervals. Lipoxygenase genes are known to be
associated with the Populus response to both abiotic and biotic
stressors (Cheng et al., 2006; Ralph et al., 2006). They are often
upregulated in the presence of mechanical damage, fungal pathogen
invasion, and exposure to simulated insect feeding (F. Chen, Liu,
Tschaplinski, & Zhao, 2009; Cheng et al., 2006). The lipoxygenases are
important in the formation of jasmonic acid, the signaling molecule that
upregulates plant defenses against herbivore feeding (F. Chen et al.,
2009).
The M. vagabunda QTL interval on Chr05 also contained a tandem
array of resistance genes (R-genes) that encoded disease resistance
proteins (TIR-NBS-LRR class) that was greatly expanded in P.
trichocarpa compared to P. deltoides , as well as repeats of the
leucine-rich repeat protein kinase family proteins in both species.
These protein families are well known for their roles in the recognition
and upregulation of host plant defenses against bacterial and fungal
infection (Bergelson et al., 2001; Martin, Bogdanove, & Sessa, 2003).
Diversification of their function through tandem duplication due to
gene-for-gene coevolution has also been demonstrated in many
plant-fungal pathosystems (Leister, 2004). Although R-genes have not
been recognized as frequently in plant-insect interactions, those that
have been discovered typically mediate interactions with
piercing-sucking insects such as aphids and whiteflies (Kaloshian,
2004).
In addition to the R-genes, there were a series of genes encoding
cytochrome P450 family proteins in both species as well as a unique
tandem set that was only present in the P. trichocarpa genome.
P450 enzymes are important in the production of many classes of
secondary metabolites such as furanocoumarins and terpenoids which are
highly toxic to insects (Keeling & Bohlmann, 2006; Schuler, 2011).
Alternatively, cytochrome P450’s may also be implicated in the
susceptibility of the host plant to galling aphids. They are important
in the synthesis of fatty acids and production of suberin in plant
tissues (Höfer et al., 2008; Pinot & Beisson, 2011). Typically, suberin
is important in separation of different tissues as well as in the
establishment of apoplastic barriers that restrict nutrient/water loss
as well as pathogen invasion (Höfer et al., 2008; Qin & LeBoldus,
2014). Several insects are known to produce suberized spherical galls on
leaves including hymenopteran pests of Rosacea species and dipteran
pests of Fabaceae (Krishnan & Franceschi, 1988; Oliveira et al., 2016).
If aphids induce the suberization mechanism of the plant genome it may
lead to the increased toughening of aphid galls, much like the woody
structures M. vagabunda leaves behind on branches once they have
finished feeding.
We detected two QTL for Phyllocolpa sp. with P.
trichocarpa alleles being positively associated with leaf fold
occurrence in both cases. The P. trichocarpa genomic interval
corresponding to the Chr10 QTL was enriched for several domains and GO
terms that could be involved in gall development. For example, the
interval was enriched for sugar transporters as well as GO terms for
sucrose transporter activity. Often in the case of galling insects plant
tissue is modified in such a way as to act as a sugar sink, thereby
enhancing its nutritional value to larvae (Larson & Whitham, 1991;
Nyman, Widmer, & Roininen, 2000; Wool, 2004) . The presence of these
combinations of sugar transporter genes may be mediating a similar
interaction between the Phyllocolpa sp. female sawflies and their
chosen Populus hosts.
Phyllocolpa sp. galls are formed early in the season when a
female sawfly selects a leaf and injects the longitudinal fold with
small amounts of fluid on the underside of young leaves (Fritz & Price,
1988; Kopelke, 2007). The adult sawfly will proceed to oviposit near the
base of the leaf and after 1-2 days the leaf fold forms and the newly
hatched larvae feeds on the inside of the gall (Smith & Fritz, 1996).
The Phyllocolpa sp. galls were a unique biotic phenotype to this
study as they were an estimate of female sawfly ovipositional choice
rather than feeding success. Host selection for oviposition is initially
driven by visual cues and reinforced by females assessing the nutrition
and chemical cues of foliage (Boeckler, Gershenzon, & Unsicker, 2011;
Panda & Khush, 1995). Previous research in Salix (closely
related to Populus ) has shown that common phenolic glycosides in
leaf tissue are important in host choice in both the free-feedingNematus oligospilus and galling Euura amerinae specialist
sawflies (Fernández et al., 2019; Kolehmainen, Roininen,
Julkunen-Tiitto, & Tahvanainen, 1994). We therefore pursued QTL mapping
of metabolites to determine if phenolic compounds might be important
determinants of the potential ovipositional relationship.
We detected three significant QTL for gentisyl alcohol 5-O-glucoside
levels. One of these overlapped with the Phyllocolpa sp. leaf
fold QTL on Chr10. The QTL on Chr14 also overlapped with a suggestive
QTL peak for Phyllocolpa sp . (LOD=3.96). This is the first case
of an association of gentisyl alcohol 5-O -glucoside with a
specialist arthropod in Populus , and is, in fact, the first
report of this metabolite in Populus . Salirepin (gentisyl alcohol
2-O -glucoside), a closely related metabolite, is a well-known
constituent in Populus sp, (Busov et al., 2006; Tschaplinski et
al., 2019; Veach et al., 2018) and leaves of P. deltoides andP. trichocarpa x deltoides have a lower abundance, later eluting
metabolite with a nearly identical fragmentation pattern to salirepin
that we tentatively identify as gentisyl alcohol 2-O -glucoside.
The QTL contains three putative candidate genes, including an aldehyde
dehydrogenase 5F1 (Podel.10G175800) that may be involved in the
reduction of gentisyl aldehyde to the alcohol, and two
UDP-glycosyltransferases (Podel.10G184800, Podel.10G185000) that may be
involved in the gentisyl alcohol conjugation to glucose. Specific
substrates have yet to be determined for these genes, but a previous
report suggests aldehyde dehydrogenase 5F1 genes are likely involved in
the basic metabolism
of Populus (Tian et al., 2015).
Phyllocolpa sp. sawflies are considered a keystone species as the
abandoned or unused leaf folds are often used as a habitat for many
other species such as aphids and spiders (Bailey & Whitham, 2007). The
presence of folds in aspen forests is associated with a two-fold
increase in arthropod species richness and around a four-fold increase
in arthropod abundance relative to forests where the insect is absent
(Bailey & Whitham, 2003). This in turn makes the host plant and sawfly
relationship important in examining how shifts in the genes of a
population ultimately structure whole communities, effectively linking
ecology and evolutionary biology. Further investigation of this
potential relationship could be key to connecting Populusgenetics to the assemblage of the surrounding communities of organisms.
A striking finding in this study was enrichment of recent tandem
duplications in the P. deltoides genome but not the P.
trichocarpa genome for the biotic QTL intervals. Out of the six
chromosomes that yielded significant QTL results, four were associated
with phenotypes that were fungi and insects native to the distribution
of P. deltoides , but not P. trichocarpa . Given thatP. deltoides has been co-evolving with the majority of the
surveyed fungi and insects, it was not unexpected that there were more
recent tandem duplicates in biotic intervals in its genome as there is
more selective pressure on the native species to overcome biotic stress
(Constabel & Lindroth, 2010; George Newcombe, Martin, & Kohler, 2010).
However, given the high amount of novel resistance occurring in the
progeny, recent tandem duplication may also be important in naïve host
resistance.
Our study has demonstrated that many recent tandem duplications, found
across biotic stress QTL intervals, have functional annotations that are
involved in physical, chemical, and tolerance mechanisms of host plant
resistance as well as a few that may be implicated in host plant
susceptibility. The enrichment of recent tandem duplications may be a
signature of gene-for-gene interactions, and a mechanism that protects
long-lived plants such as trees, enabling them to reach maturity despite
many coevolving biotic stressors. In addition to the direct effect of
the host plant genetics on associated organisms, we have also
demonstrated an indirect community effect that was mediated by thePopulus progeny (Whitham et al., 2006). The fungal pathogen
succession highlights the complexities of how host plant-pathogen
coevolution can affect multiple species interactions in more subtle ways
than expected in a natural environment.