Document 1949103

Candidate genes for resistance and susceptibility to
the bird cherry-oat aphid (Rhopalosiphum padi L.) in
barley (Hordeum vulgare L.)
Sara Mehrabi
Candidate genes for resistance and
susceptibility to the bird cherry-oat
aphid (Rhopalosiphum padi L.) in
barley (Hordeum vulgare L.)
Sara Mehrabi
©Sara Mehrabi, Stockholm University 2016
Cover photo: Sate Al Abbasi
ISBN at final page
Printed in Sweden by Holmbergs, Malmö 2016
Distributor: Department of Ecology, Environment and Plant Sciences
Stockholm University, Stockholm, Sweden
“Dedicated to my beloved parents”
Sammanfattning
Havrebladlusen (Rhopalosiphum padi L.) är en viktig skadegörare i vårsäd.
Angrepp av havrebladlus ger inte synliga symptom men svåra angrepp kan
orsaka allvarlig skördeminskning och bladlusen kan överföra virus som orsakar rödsot. Havrebladlusen är en av de allvarligaste skadegörarna på korn,
som produktionsmässigt är den fjärde grödan globalt sett. För att hantera
havrebladlus används bekämpningsmedel. Nackdelar med detta är att flera
av dem orsakar negativa effekter i miljön och att tillstånden för användning
riskerar att dras in. En annan nackdel är att bekämpningsmedel inte är långsiktigt verksamma på grund av att bladlöss utvecklar resistens mot dem. Ett
alternativ för att hantera problemet med havrebladlus är att identifiera genetiska faktorer som kan användas i resistensförädling. Det finns många exempel på framgångsrik förädling för bladlusresistens, men det finns ännu ingen
kornsort som är resistent mot havrebladlus. Syftet med denna avhandling var
att identifiera och karakterisera faktorer i korn som ger resistens mot eller
ökad mottaglighet för havrebladlus. Detta gjordes med två huvudsakliga
angreppssätt. I det första studerades uttrycksnivåer av utvalda gener i ett
stort antal förädlingslinjer av korn som var karakteriserade vad gäller resistens mot havrebladlus. Genuttrycket studerades både med och utan havrebladlöss på plantorna. Den andra metoden var att isolera cDNA som förmodades koda för bladlusresistens från korn och överföra det för uttryck i modellväxten backtrav (Arabidopsis thaliana L.) och starkare uttryck i korn.
Effekterna av genöverföring utvärderades med hjälp av biologiska test med
bladlöss.
Studierna av genuttryck på plantor utan bladlöss i 23 olika genotyper av
korn visade att tre olika gener möjligen är relaterade till resistens eller mottaglighet mot havrebladlus. De två gener som visade starkare uttryck i resistenta linjer kodar för ett svavelinnehållande protein (tionin) samt för en
hämmare av proteaser. Den gen som hade starkare uttryck i mottagliga linjer
kodar för ett lipoxygenas. I studierna av genuttryck i plantor med bladlöss
studerades särskilt tre glukanaser. Resultaten gav stöd för idén om att två av
dessa kan bidra till mottaglighet för havrebladlus.
cDNA för protashämmaren överfördes till backtrav med kontroll av två olika
promotorer, varav den ena ger genuttryck i all växtvävnad och den andra
särskilt i floemvävnad. Havrebladlus kan inte överleva på backtrav, och testen utfördes därför med persikbladlus (Myzys persicae Sulzer). Persikbladlus
kan till skillnad från havrebladlus livnära sig på många olika arter, både tvåhjärtbladiga (som backtrav) och enhjärtbladiga (som korn). Resultaten visade
att persikbladlusen föredrog att äta från och producerade större antal avkomma på kontrollplantor jämfört med flera av linjerna som uttryckte genen
för proteashämmaren.
Sammanfattningsvis ger denna avhandling stöd för idéer om att ett tionin
och en proteashämmare kan vara involverade i resistens mot havrebladlus
och att ett lipoxygenas och två glukanaser kan påverka mottagligheten för
havrebladlus positivt. Studien visar vidare att cDNA från korn som valts ut
på grund av att det induceras i en enhjärtbladig växt av en tämligen specialiserad bladlus kan påverka resistensen mot en helt annan bladlusart i en tvåhjärtbladig växt.
List of papers
This thesis is based on the work contained in the following papers and manuscripts, referred to in Roman numerals in the text:
I. Mehrabi S, Åhman I, Jonsson LMV (2014) Transcript abundance of
resistance- and susceptibility related genes in a barley breeding pedigree
with partial resistance to the bird cherry-oat aphid (Rhopalosiphum padi L.).
Euphytica 198:211-222.
II. Mehrabi S, Åhman I, Jonsson LMV (2016) The constitutive expression
and induction of three ß-1,3-glucanases by bird cherry-oat aphid in relation
to aphid resistance in 15 barley breeding lines. Arthropod-Plant Interactions
doi:10.1007/s11829-016-9415-2.
III. Losvik A, Beste L, Mehrabi S, Jonsson LMV. An aphid-induced
proteinase inhibitor from barley affects aphid performance in transgenic
Arabidopsis (Manuscript).
IV. Mehrabi S, Beste L, Stephens J, Jonsson LMV. Aphid performance on
Arabidopsis and barley overexpressing an open reading frame, up-regulated
in Rhopalosiphum padi resistant barley lines during aphid infestation. (Manuscript).
Paper I and II are reproduced with the permission of their respective publisher.
My contributions to the papers:
Paper I: Participated in the experimental design, performed the experiments
and the analysis of the data. Contributed to writing the paper.
Paper II: Was main responsible for the experimental design, performed the
experiments and the analysis of the data. Contributed to writing the paper.
Paper III: Participated in the experimental design and in performing the
experiments. Commented on the manuscript.
Paper IV: Was main responsible for the experimental design, performed the
experiments and the analysis of the data, except for cloning and transformation. Wrote the paper in cooperation with co-authors.
Contents
General introduction ..................................................................................... 12 Barley – an ancient agricultural important crop ............................................................ 12 Aphids as pests ............................................................................................................ 13 General background ................................................................................................ 13 Aphid damage to plants .......................................................................................... 14 Bird cherry-oat aphid (R. padi) ................................................................................ 15 Pest management ................................................................................................... 16 Resistance breeding ..................................................................................................... 17 Constitutive and induced plant defense .................................................................. 17 Morphological and surface barriers ......................................................................... 18 Secondary metabolites ............................................................................................ 19 Induced defense mechanisms and different types of resistance ............................ 20 Breeding methods ................................................................................................... 22 Plant material used in the study .............................................................................. 23 Main approaches in this thesis ..................................................................................... 25 Constitutive gene expression in a large number of barley genotypes with known
resistance levels to R. padi (Paper I) ...................................................................... 25 Aphid-induced gene expression of putative susceptibility factors in a large number
of barley genotypes with known resistance levels to R. padi (Paper II) .................. 25 Aphid behavior and performance on transgenic Arabidopsis with a putative aphid
resistance sequence from barley (Paper III) ........................................................... 27 Aphid behavior and performance on transgenic Arabidopsis and barley with a
putative aphid resistance sequence from barley (Paper IV) ................................... 29 Main results and discussion ......................................................................... 31 General discussion about methods.............................................................. 33 Conclusions ................................................................................................. 37 Suggestions for further studies .................................................................... 38 Acknowledgements ...................................................................................... 39 References ................................................................................................... 40 Abbreviations
BC
BCI
BYDV
Col-0
F1
HAMPs
Hsp5
JA
LOX
MAMPs
NBS-LRR
ODN
ORF
PAMPs
PI
PI (s)
QTL
qPCR
R
RNAi
RNA-seq
S
SA
STK
THIO
VIGS
ßGLU-GIII
Back-cross
Barley Chemically Induced
Barley yellow dwarf virus
Columbia
First generation of offspring after crossing
Herbivore-associated molecular patterns
Hordeum vulgare subspecies spontaneum accession 5
Jasmonic acid
Lipoxygenase
Microbe-associated molecular patterns
Nucleotide-binding site leucine-rich repeat
Oligodeoxynucleotides
Open reading frame
Pathogen-associated molecular patterns
Proteinase inhibitor (paper I) = BCI-7 (paper III)
Proteinase inhibitor (s)
Quantitative trait locus
Quantitative polymerase chain reaction
Resistant
RNA-mediated interference
RNA sequencing
Susceptible
Salicylic acid
Putative serine/threonine kinase sequence (paper I) =
(contig16360_at, paper IV)
Thionin
Virus induced gene silencing
beta-1,3-glucanase III (paper 1) = (contig1636_at, paper II)
General introduction
The overall objective of this thesis was to identify genes related to resistance
or susceptibility towards the bird cherry-oat aphid (Rhopalosiphum padi L.)
in barley (Hordeum vulgare L.).
Aphids are a major threat to crop production reducing the yield if not
controlled. R. padi attacks all small grain cereals including e.g. wheat,
triticale and barley (Blackman and Eastop 2007). According to the Food and
Agriculture Organization of the United Nations statistics in 2013, barley had
the fourth rank in the world cereal production after maize, rice and wheat
(FAOSTAT 2013). Consequently, sustaining high yields of barley under
conditions of abiotic and biotic stresses is needed (Maheswari et al. 2012).
Aphids are the most important insect pests on barley and damage the
plants both directly by feeding and indirectly by transmitting viral diseases
(Mornhinweg 2010). Chemical control is the most efficient and widely used
management tool to control aphids but using insecticides has negative
environmental consequences and causes insecticide resistance in the aphids.
As an example, resistance against the major insecticides has developed in the
green peach aphid (Myzus persicae Sulzer) worldwide (Radcliffe et al.
2007).
An alternative to the chemical control of aphids is to breed for aphid host
plant resistance. The work in this thesis aims to identify resistance or susceptibility factors in barley. This is done by the study of constitutive and
induced gene expression in barley genotypes with different degree of
resistance against R. padi, and by transformation of candidate resistance
sequences and evaluating their effects on aphids in barley and Arabidopsis.
Barley – an ancient agricultural important crop
Barley belongs to the tribe Triticeae and the family Poaceae. Hordeum
vulgare ssp. spontaneum (wild barley) is considered as the ancestor of cultivated barley (Hordeum vulgare ssp. vulgare) (von Bothmer and Komatsuda
2010). There are two- and six-rowed barley varieties as well as winter and
spring types. Archaeological evidence shows that the domestication of
barley started about 10,000 years ago in what is called “the fertile crescent”,
that is the region in the Middle East, with a curved shape from the Persian
Gulf to the Mediterranean Sea. Barley is a diploid (2n=14), self-pollinated
12
plant with a simpler genome compared to the hexaploid wheat and it has
been argued that it is a very good model organism in molecular research for
bread wheat (Schulte et al. 2009). The barley genome was shotgun
sequenced recently and its genome size was reported to be about 5.1
gigabases and contain 85% repetitive elements (International Barley Genome Sequencing Consortium 2012). This advancement is a significant help
to develop new cultivars of barley, which are able to withstand pests and
diseases.
Barley is used as animal feed, in beverage production and as human food.
Barley is valuable in the production of malt that is mainly used in the beer
and beer-like beverage industry and to a lesser extent in baking, distilled
beverage production, fuel (ethanol) and confectionary industry. As a whole
grain food, various studies have put forward positive health effects, i.e. that
the consumption could reduce blood cholesterol (Åman 2006), and blood
glucose (Beta et al. 2012).
Barley is attacked by more than 100 arthropod pests and many of them
cause economic damage in outbreak years, such as Russian wheat aphid
(Diuraphis noxia Mordvilko), bird cherry-oat aphid (R. padi) and greenbug
(Schizaphis graminum Rondani). Other serious pests on barley are the army
cutworms (Euxoa auxiliaris Grote), grasshoppers (Melanoplus sanguinipes
Stål) and wireworms (Agriotes species) (Mornhinweg 2010).
Aphids as pests
General background Aphids are important and common pests in temperate regions. They are
members of the superfamily Aphidoidea, suborder Sternorrhyncha, order
Hemiptera. There are ca. 4700 aphid species, but only about 100 of them are
considered as economically important on crop plants (Blackman and Eastop
2007). Aphids have soft bodies, vary in size between 1 to 10 mm and have
different colors (yellow, green, red or black). They have slender piercingsucking mouthparts and cornicles on the abdomen. The cornicles are tubelike structures, which release alarm pheromones after attack by natural
enemies of aphids (Gullan and Martin 2009).
Aphids feed on plant phloem sap. They penetrate the epidermis with their
stylet and push it through the parenchymatic tissue extracellularly until they
reach the vascular bundles of the plant (Fig. 1). When the aphid stylet
reaches the phloem, the aphid can feed on phloem sap for several hours and
even days (Tjallingii 1995). As soon as an aphid stylet enters between the
borders of two epidermal cells, saliva is secreted that jellifies and facilitates
the stylet´s penetration of plant tissue. Watery saliva is released as the stylet
13
reaches the sieve element. This saliva has been shown to suppress protein
clogging which would otherwise inhibit phloem transport and aphid feeding
(Tjallingii 2006; Will et al. 2009). Aphid feeding causes the nutrients to flow
into the aphid-infested tissue. As a consequence, the flow of nutrients into
other sink areas such as the primary growth zone will be reduced (Mittler
and Sylvester 1961). The phloem sap is very rich in sucrose and poor in
amino acids, which are the main essential nutrients for aphids. Aphids cannot synthesize all their essential amino acids and are dependent on endosymbiotic bacteria in order to obtain those (Shigenobu et al. 2000). Occasionally
aphids also ingest xylem sap.
Fig. 1 Aphid feeding on phloem sap. st: stylet, scl: sclerenchyma, p: phloem, x:
xylem. Scale bar = 1 mm. The picture is adapted from Plants in Action, published by
the Australian Society of Plant Scientists
(http://plantsinaction.science.uq.edu.au/edition1).
Aphid damage to plants
Most of the crops in the world are attacked by at least one aphid species.
Aphids cause asymptomatic (not visible) or symptomatic (visible) injury.
Some species, including R. padi on cereals, pea aphid (Acyrthosiphon pisum
Harris) on legumes and soybean aphid (Aphis glycines Matsumura) on
soybean, do not cause visible damage but they can reduce plant growth. In
contrast to the previous group, some species cause visible damage on plants
including the Western wheat aphid (Diuraphis tritici Gillette) that causes
14
stunting, D. noxia that causes chlorosis, leaf rolling and stunting and
S. graminum that causes necrosis in cereals (Quisenberry and Ni 2007).
Aphids are well-known as plant virus vectors and over 190 aphid species
have been described as viral vectors (Katis et al. 2007). The barley yellow
dwarf virus (BYDV) is a worldwide virus, infecting important cereals causing dwarfing and leaf discoloration. In years with heavy aphid infestation,
BYDV leads to severe yield losses in cereals. Worldwide, most barley cultivars are susceptible to BYDV and the virus may reduce the plant yield by up
to 46% (Schliephake et al. 2013). In addition to the above negative consequences, the sugary honeydew accumulating on leaves infested by aphids
promotes fungal growth. Thereby, growth of saprophytic fungi can result in
blocking of the stomata, reduction of the photosynthesis and earlier shedding
of leaves (Morkunas et al. 2011).
Bird cherry-oat aphid (R. padi)
R. padi is one of the most serious pests on cereal crops and one of the five
most important barley pests worldwide (Mornhinweg 2010). Its color is
varying from green-olive to yellow-green, dark olive or greenish-black with
a red-orange pigmentation around the bases of the posterior part of the
abdomen in the apterous (wingless) individuals. R. padi can have an
holocyclic life cycle with cyclic parthenogenetic reproduction on both a
primary (winter) and a secondary (summer) host; or an anholocyclic life
cycle with obligate parthenogenetic reproduction all year round on the
secondary host in areas where the primary host is rare or during mild winters
(Simon et al. 1996).
R. padi with holocyclic life cycle (Fig. 2) passes the winter in the form of
eggs on bird cherry (Prunus padus L.). The eggs hatch in spring and after
some apterous generations on the primary host, winged aphids develop that
migrate to grasses (secondary hosts). Several apterous generations are born
on the summer host. As the density of aphids increases or the nutrient source
is depleted, winged aphids develop that migrate to new summer hosts. With
the approach of autumn, winged females and males are born. The winged
females (gynoparae) give birth to apterous egg-laying forms (oviparae) that
mate with winged males and lay eggs on the winter hosts (Dixon 1971). As
mentioned above, this aphid does not cause visible damage, but at high
infestation rates, it can reduce the growth and yield by removal of plant
nutrients (Dixon 1971; Mornhinweg 2010). It is also an efficient vector of
most small grain viruses (e.g. BYDV) worldwide (Blackman and Eastop
2007, Mornhinweg 2010).
15
Bird'cherry'
Grass'
Summer'
Winter'
Spring'
Autumn'
Fig. 2 R. padi holocyclic life cycle. Fundatrix is an apterous female that parthenogenetically (non-sexually) produces live offspring. Gynopara is a female winged
aphid that migrates to the primary host and produces oviparae. Ovipara is an
apterous sexual female that mates with a winged male aphid and lays eggs (Figure
inspired by Dixon 1971).
Pest management
Insecticides are used extensively to control aphid infestation. However, there
are disadvantages with this method, such as negative effects on the
ecosystem, notably on non-target beneficial insects (predators, parasitoids
and pollinators) and the risk of aphids developing resistance to the insecticides. There is a long list of pesticides active against aphids and new
aphicides are being developed worldwide. In the 1990s, mostly
organophosphates, carbamates and pyrethroid insecticides were used to control aphids and some of these insecticides have high toxicity to non-target
insects (Dewar 2007). In the European Union, the regulations for the application of pesticides are becoming increasingly restrictive and e.g. in Sweden
at
16
present there are few products available for farmers (Gustafsson 2015;
Bekämpningsmedelsregistret 2016).
Resistance breeding
In view of the above situation, there is an urgent need to develop alternative
ways to fight aphids, such as breeding for host plant resistance. Growing
cultivars that are insect-resistant is an environmentally friendly, efficient and
easy to use method for the farmer. The resistant traits can be classified as
antixenosis, antibiosis and tolerance traits. Antixenosis or non-preference is
when the herbivore avoids feeding or reproducing on the plant. Antibiosis is
the negative effect of plant biology on pest physiology. It can result in the
reduction of insect life span and reproduction as well as increasing the insect
mortality. Tolerance is a mechanism that enables plants to withstand the
herbivore, and recover from its damage and thus maintain biomass and yield
production. The difference between antixenosis or antibiosis and tolerance is
that the first two types of resistance measure the response of the insect to the
plant, whereas tolerance measures the response of the plant to the insect
(Smith 2005).
Constitutive and induced plant defense
When searching for resistance factors to be used in breeding, it is important
to know the inherent defense mechanisms in plants. Although plants have a
sedentary lifestyle, they have a dynamic interaction with insects (Mello and
Silva-Filho 2002; Mescher and De Moraes 2015). Plants have both constitutive and inducible defense strategies against insect herbivores (Fig. 3).
Constitutive defenses are including physical and chemical barriers that exist
before insect attack, whereas induced defenses become active by insect
attack. Induced plant defenses can be either direct or indirect. Direct
defenses are the plant traits that affect herbivore biology directly, e.g.
delaying their development or even killing them. Indirect defenses are when
e.g. plants volatiles are induced which attract other organisms that may aid
to suppress the growth of the herbivores (War et al. 2012; Fürstenberg -Hägg
et al. 2013).
17
vola6les"
Epidermis"
Mesophyll"
Companion"cell"
Phloem"
Gene"ac6va6on"
Direct"responses"""""""""""""""""""""""""""
Indirect"responses"""""""""""""""""""""""""""
!"Physical"barriers"
"
!"Secondary"metabolites"
"
!"Inhibitors"
"
!"Proteins"
"
Fig. 3 Generalized scheme of plant defense reactions to insects, with aphids
and ladybirds as examples of herbivores and insect natural enemies (Figure
inspired by Mello and Silva-Filho 2002).
Morphological and surface barriers
Plant leaves and stems may carry trichomes, which can constitute a physical
barrier against insects. Certain trichomes contain essential oils and other
secondary metabolites that act as chemical defense (Wagner et al. 2004).
Trichomes have been much studied in the context of insect defense and
among them are some studies on their relevance in aphid defense.
The results suggest that the effects of trichomes vary depending on the plant
and aphid species. There are reports of negative effects of trichomes on
aphid performance in wild species of Lycopersicon against M. persicae
(Simmons et al. 2005) and against melon aphid on melon (Sarria et al. 2010),
but there are also reports of no effects of trichome density in soybean upon
the abundance of A. glycines (Dai et al. 2010). In barley, R. padi survival and
nymphal growth was studied in a number of breeding lines of which some
were densely hairy and others not at all hairy. There was no difference in the
aphid parameters between these categories of lines, indicating that hairs are
not important in barley against R. padi (Åhman et al. 2000).
The wax and cutin layers of the leaf are also considered as structural layers of defense. These components have been shown to reduce aphid population growth in some cases. As an example, English grain aphid (Sitobion
avenae Fabricius) had longer non-probing periods on waxy compared to
18
waxless triticale plants and surface waxes from waxy plants had aphicidal
activity against S. avenae (Wójcicka 2015).
Secondary metabolites
Plant secondary metabolites may be part of the defense against insect herbivores. Such metabolites accumulate constitutively or are induced after
pathogen or herbivore attack. They may have different activities including
toxicity, deterrence and repellence (Bennett and Wallsgrove 1994). Among
the wide variety of secondary metabolites found in the plant world, there are
some that have been found to play a role in the interaction between plants
and aphids.
A basic idea concerning the role of secondary metabolites and plant
defense is that they are toxic to generalist insects but that specialized insects
have found ways to overcome their toxicity and use them to their own favor.
Such insects may even need them as feeding or oviposition stimuli. This is
nicely illustrated in the case of aphids on lupines (Lupinus angustifolius L.).
Lupines contain quinolizidine alkaloids, but the contents vary between the
cultivars. Generalist aphids, including M. persicae, feed on sweet lupines,
which are almost alkaloid-free varieties, but avoid alkaloid-rich varieties. In
contrast, the specialized lupine aphid (Macrosiphum albifrons Essig) is well
adapted to lupines with high alkaloid content. This aphid stores the alkaloids
and uses them for its defense against predators (Philippi et al. 2015). Similar
relations have been shown in the case of the glucosinolates in the Brassicaceae and their associated aphids.
Glucosinolates are secondary metabolites restricted mainly to the Brassicaceae family. Upon tissue damage, glucosinolates are hydrolyzed by
myrosinases into products, including isothiocyanates, which have toxic and
deterrent effects on some herbivores (Halkier and Gershenzon 2006). There
is much evidence that these compounds are deterrents and repellents towards
generalist insects but may act as attractants or feeding and oviposition stimulants to specialist insects, including aphids (Bruce 2014). The specialist
cabbage aphid (Brevicoryne brassicae L.) was shown to sequester
glucosinolates from its host and to compartmentalize myrosinase into
microbodies in the muscles, which in combination might help this aphid to
defend itself against predators (Bridges et al. 2002).
Benzoxazinoid hydroxamic acids constitute another group of secondary
metabolites and are present in cereals including maize, wheat, rye and wild
Hordeum species but not found in cultivated barley (Niemeyer 2009). There
are reports of correlations between the performance of cereal aphids and the
content of benzoxazinoid hydroxamic acids in different wheat cultivars (Argandoña et al. 1980). Clear negative effects of the benzoxazinoid 2, 4dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) in maize
have been reported on R. padi (Ahmad et al. 2011) and corn leaf aphid
19
(R. maidis Fitch) (Meihls et al. 2013), using genetically well-defined maize
lines.
With regard to barley there are some studies on the indole alkaloid
gramine indicating that it is a resistance factor against cereal aphids (Zúñiga
et al. 1985; Zúñiga and Corcuera 1986). However later investigations raised
doubt on the role of gramine in barley resistance to R. padi by showing no
relationship between gramine content and settling preference (Forslund et al.
1998) or aphid growth (Åhman et al. 2000) in two different large selections
of barley genotypes.
Induced defense mechanisms and different types of resistance
Induced defense mechanisms have the advantage that they are only
expressed when the plant is attacked. Thus, there will be no yield penalty
under the conditions that there is no aphid infestation.
Current models of plant responses to pathogens and other attackers suggest that plants have two types of defense responses, basal defense and
resistance (R) gene mediated defense (also called effector-triggered immunity) (Chisholm et al. 2006; Jones and Dangl 2006; Bent and Mackey et al.
2007).
In the basal defense, the plant recognizes pathogen associated molecular
patterns (PAMPs), microbe associated molecular patterns (MAMPs) or
herbivore associated molecular patterns (HAMPs) by plant pattern recognition receptors that result in the activation of PAMP/MAMP/HAMP-triggered
immunity.
PAMPs/MAMPs/HAMPS activate basal early defense responses that take
seconds to minutes (e.g. ion flux and oxidative burst), intermediate defense
responses that take minutes to hours (e.g. kinase cascade activation) and late
responses that take hours to days, e.g. salicylic acid (SA) accumulation and
callose deposition (Zipfel and Robatzek 2010). There are similarities in signaling pathways in plant-pathogen and plant-herbivore interactions including
ion fluxes, kinase cascade activations, phytohormone generation, activation
of defense-related genes and synthesis of secondary compounds and volatiles
that suggest similar recognition processes in plant-herbivore and plantpathogen interactions (Mithöfer and Boland 2008).
Aphid feeding on plants activates both the SA- and the jasmonic acid
(JA) -mediated signal pathways (Morkunas et al. 2011; Louis et al. 2012). It
has been suggested that efficient plant defence against phloem-feeding insects is regulated by JA (Walling 2008). There is support for this notion, but
there are also reports of aphid resistance related to SA-induced responses
(Morkunas et al. 2011; Avila et al. 2012; Louis et al. 2012).
Successful pathogens or herbivores answer by sending effectors into the
plant that inhibit the defense reactions. In the case of aphids, several potential aphid salivary effector proteins have been identified that can modulate
20
plant defense, but only few of them are functionally characterized (Wang et
al. 2015). Several studies were done on the C002 protein, which is aphidspecific, and appears to have a role as a salivary effector protein. RNAisilencing or overexpression of the sequence coding for the C002 protein in
Nicotiana benthamiana (Domin) decreased aphid fitness or increased aphid
reproduction, respectively (Elzinga et al. 2014). Expression of the sequences
for two candidate effectors, Me10 and Me23, from potato aphid
(Macrosiphum euphorbiae Thomas) in N. benthamiana increased aphid
fecundity (Atamian et al. 2013). A recent study by Wang et al. (2015) suggested that a protein named Armit is another aphid salivary effector protein.
In R-gene mediated defense, an R-protein recognizes the effector(s) (also
called avirulence (Avr) gene products) from pathogens or pests. This results
in effector-triggered immunity and stronger defense (Fig. 4) (Kaloshian
2004). Plant R-genes are classified into 8 groups based on the membrane
spanning domain and amino acid motif organization (Gururani et al. 2012).
Most of the known R-genes in plants encode nucleotide-binding site leucinerich repeat (NBS-LRR) proteins and provide resistance to pathogens and
insect herbivores (Jones and Dangl 2006; Hogenhout and Bos 2011).
To date, two aphid R- genes have been isolated. The Mi-1.2 gene was
isolated from a wild relative to tomato and confers resistance against certain
biotypes of M. euphorbiae. The Vat gene was found in melon and confers
resistance to the melon-cotton aphid (Aphis gossypii Glover) (Smith and
Chuang 2014). They both belong to the NBS-LRR family of resistance
genes.
Other terms for R-based resistance are qualitative or vertical resistance,
indicating that it gives a very strong specific resistance. The disadvantage is
that this type of resistance is only triggered in the interaction with attackers
containing avirulence gene products (effectors). Thus, the resistance is not
efficient against aphid biotypes without the avirulence protein (Fig.4) (Poland et al. 2009).
Another type of resistance is called horizontal or quantitative. This type
of resistance is controlled by several genes each of them adding to the
resistance. This type of resistance is more durable, but has as disadvantages
that the resistance is not so strong and that it is more difficult to identify the
genes adding to the resistance and more difficult to breed for (Poland et al.
2009). The present thesis is focusing on finding genes involved in
quantitative resistance.
21
Rsistance
Aphid Fig. 4 Schematic picture of R-gene mediated defense in the interaction between a
plant and aphids. HR = hypersensitivity response, often associated with this type of
resistance. Picture modified from Gururani et al. (2012).
Breeding methods
To introduce desirable traits into a plant, conventional and transgenic breeding methods may be used. Conventional breeding means the crossing
between parental lines and progeny selection. The isolation of homozygous
lines carrying the desirable traits might need several rounds of backcrossing, inbreeding and selection, which needs much time and labor.
To improve and shorten this process, the methods of marker-assisted breeding and doubled haploid production have been developed.
In a breeding program the superior plants are selected by evaluating the
breeding population visually (phenotypic selection) in field trials (e.g.
resistance or agronomic traits) or by chemical tests (e.g. grain quality),
which needs considerable amounts of time (Collard and Mackill 2008).
Marker-assisted selection technology makes it possible to select the superior
plants by use of a DNA marker that is linked to the gene of interest and can
accelerate the breeding process. In comparison to phenotypic selection,
marker-assisted selection saves time, resources and effort, can be carried out
at seedling stage and a single plant can be selected based on its genotype. In
breeding for R. padi resistance, this method would be very useful, since the
phenotyping methods require very laborious bio-assays based on aphid
growth, development and behavior.
Doubled haploid production includes the cultures of plants from single
pollen or microspores (Friedt et al. 2010) and is used in many crops, for example barley, wheat (Triticum aestivum L.), maize (Zea mays L.) and rapeseed (Brassica napus L.) (Li et al. 2013). By doubled haploid production,
complete homozygosity can be obtained in one generation, while it may
otherwise take six to eight generations to achieve almost full homozygosity,
about three to five years, depending on the crop (Friedt et al. 2010).
22
Transgenic breeding is a method for transferring desirable agronomic
traits into other species or changing the level of expression of available traits
in a species. This method has the advantage that one very specific and desirable trait may be changed, whereas using conventional methods of crossing,
many undesirable traits are likely to be transferred.
Several aphid-resistant cultivars have been developed by conventional
breeding during the last decades in important agricultural and horticultural
crops including maize, sorghum, tall fescue, barley, wheat, soybean, barrel
medic, lettuce, tomato, apple, melon, peach, peanut, pear, red raspberry,
black raspberry (Smith and Chuang 2014). The resistances are either known
to be based on classical resistance (R-) genes, or have been mapped to chromosome regions encompassing NBS-LRR sequences, thus with the disadvantage that the resistance may be overcome. So far biotypes virulent to
cultivars with R-genes have been found in 17 aphid species. This illustrates
the constant need of developing new cultivars with aphid resistance (Smith
and Chuang 2014). It has also resulted in an increased interest in searching
for quantitative resistance, based on several traits, which is also the focus in
this thesis.
Plant material used in the study
The barley plant material used in this thesis was a selection of barley
breeding lines and their parents, which had been characterized with regard to
R. padi resistance. The breeding lines had been obtained from a cross
between the moderately R. padi resistant wild barley accession Hsp5
(H. vulgare ssp. spontaneum) and cultivar Lina. The first generation (F1)
line 660-6:8 was selected as a partially resistant offspring and backcrossed
with cultivar Lina and crossed with cultivar Barke that resulted in the first
backcross (BC) generations (Fig. 5). Selected resistant offspring lines from
the BC1 were further backcrossed to cultivar Lina or cultivar Barke and
resulted in BC2 lines. All the BC lines are from doubled haploids. The
genotypes were characterized as resistant or susceptible based on an assay of
nymphal growth, where 5 nymphs were released in a cage fixed at the basal
part of the plant and nymphal weight was evaluated after 4 days (Åhman et
al. 2000). The results of the bioassay correlated with quantitative trait loci
(QTLs) markers for aphid resistance (Cheung et al. 2010) and predicted well
field resistance level (I. Åhman, personal communication).
23
!
Lina%(S)%x%660,6:8%(R)%%
F1!
BC1!
BC2!
Lina%(S)%x%Hsp5%(R)%
5172%28:4%%(R)%
x%Lina%(S)%
Barke%(S)%x%660,6:8%(R)%%
5172%39:9%(R)%%
x%Lina%(S)%
5172%48:12+(R)%%
x%Lina%(S)%
5175%50:20+(R)%%
x%Barke%(S)%
6652%101%(R)%
6653%62%(R)%
6654%117%(R)%
6655%68%(R)%
6652%179%(R)%
6653%210%(R)%
6654%194%(R)%
6655%25%(S)%
6652%79%(S)%
6653%44%(S)%
6654%18%(S)%
6655%135%(S)%
6652%143%(S)%
6653%150%(S)%
6654%123%(S)%
Fig. 5 Relationships between barley genotypes used in papers I and II. All BC1 and
BC2 lines are doubled haploids (bold) and characterised as aphid resistant (R) or
aphid susceptible (S).
24
Main approaches in this thesis
Constitutive gene expression in a large number of barley genotypes with
known resistance levels to R. padi (Paper I)
In paper I, the constitutive expression of a selection of sequences was
explored in a collection of barley breeding lines with different degree of
aphid resistance. The aim was to find out if the genes had higher expression
in resistant as compared to in susceptible genotypes (or vice versa). This
approach is based on the hypotheses (a) that the resistance or susceptibility
character is to some extent constitutive and (b) that the transcript abundance
of a certain gene sequence gives information about the physiological state of
the plant.
Regarding the hypothesis (a) some examples of constitutive differences in
trichomes and secondary metabolites relating to aphid resistance have been
given above. In addition, recent literature report e.g. constitutive differences
in some enzyme activities related to defense (phenyl ammonia lyase, polyphenol oxidase and peroxidase) between S. avenae-resistant and -susceptible
wheat genotypes (Han et al. 2009) and differences in the constitutive amino
acid profiles between soybean genotypes resistant and susceptible towards
A. glycines (Chiozza et al. 2010). Constitutive differences in the transcript
abundance of a number of gene sequences were reported in a comparative
study between R. padi resistant and susceptible barley genotypes (Delp et al.
2009) and these results were the basis for the study presented here.
Regarding the hypotheses (b) a large number of studies supported the
default assumption that higher transcript abundance indicates higher protein
levels, but there are also reports that did not support the assumption. For
example, the higher transcript level of certain genes involved in the
biosynthesis of glucosinolates after Arabidopsis infestation by cabbage aphid
(B. brassicae) did not relate to glucosinolate level (Mewis et al. 2006). Also,
in barley, it was found that higher transcript abundance for a protein
involved in gramine biosynthesis, N-methyltransferase (NMT), induced by
R. padi or rose-grain aphid (Metopolophium dirhodum Walker) infestation
did not lead to higher amounts of the NMT protein (Larsson et al. 2011).
Aphid-induced gene expression of putative susceptibility factors in a large
number of barley genotypes with known resistance levels to R. padi (Paper
II)
In paper II, the constitutive and aphid-induced expression of sequences putatively related to aphid susceptibility were investigated in a collection of
barley breeding lines with different degrees of aphid resistance. The hypothesis is that certain sequences which have higher expression in susceptible
25
compared to in resistant genotypes increase susceptibility to R. padi. Breeding for loss of susceptibility is a strategy to develop durable and broadspectrum resistant cultivars. This method has proven useful in the case of the
loss of the Mlo gene function in barley, which has given resistance to
powdery mildew (Pavan et al. 2010; Zheng et al. 2013). Susceptibility genes
are those that code for proteins necessary for or helpful to the pathogen or
the pest. They are expected to be expressed in susceptible plants or to be upregulated in such plants upon infection or infestation. Based on this idea, all
gene sequences up-regulated in susceptible plants upon aphid infestation are
putative susceptibility genes and those upregulated specifically in
susceptible plants (and not resistant plants) are even stronger candidates.
Regarding the hypothesis, in several studies, gene expression and enzyme
activity were increased in susceptible plants upon aphid infestation.
There is support for the idea that ß-glucanases are susceptibility factors in
plant-aphid interactions (Botha et al. 2010; Reddy et al. 2013; Anderson et
al. 2014). In two separate comparative studies between resistant and susceptible cultivars and between tolerant and susceptible cultivars in barley,
several genes were up-regulated only in susceptible lines by R. padi and
D. noxia, respectively (Delp et al. 2009; Gutsche et al. 2009).
This study builds on paper I and an earlier study comparing transcript
abundance with and without aphids in two susceptible and two resistant
barley genotypes (Delp et al. 2009). Barley ß-1,3-glucanases were investigated as putative susceptibility factors in the barley- R. padi interaction.
ß-1,3-glucanases are one of the important groups of hydrolytic enzymes,
belonging to the pathogenesis-related proteins family 2 (Balasubramanian et
al. 2012) and their transcription increases significantly after pathogen and
insect attack in several plant species (van Loon et al. 2006). The exact function of glucanases in plant defense against aphid is unclear (van der
Westhuizen et al. 1998, Botha et al., 2014), but it may facilitate aphids
probing and feeding by the degradation of callose and cell wall structures
(Hao et al. 2008; Anderson et al. 2014).
Higher plants produce several ß-1,3-glucanases isoforms with different
primary structures and size, cellular localization and pattern of regulation.
Based on their amino acid identity they are classified as: class I, II and III.
Each of them may have a different biological function. ß-1,3-glucanases
(commonly found all over the plant kingdom) and ß-1,3;1,4-glucanases (reported only in monocots of the Poaceae family) are two structurally related
ß-glucanases which both have a role in the defense against pathogens, but
degrade different linkages in ß-glucans present in the cell wall and produce
different products (Simmons 1994). ß-1,3-glucanases recognize and cleave
ß-1,3-linkages and ß-1,3;1,4-glucanases recognize and cleave ß-1,3;1,4linkages. ß-glucan degradation releases fragments from the plant cell walls,
which have been shown to be activators of several defense related genes
including ß-glucanases (Hrmova and Fincher 2001).
26
Aphid behavior and performance on transgenic Arabidopsis with a
putative aphid resistance sequence from barley (Paper III)
In paper III and IV, the function of two putative aphid resistance related
genes in barley was examined by transformation of the respective cloned
cDNA´s into Arabidopsis and barley, followed by study of the effects on
aphid biology.
The approach in paper III is based on two hypotheses; (a) that genes upregulated by R. padi in moderately resistant barley and not in susceptible
barley may confer increased resistance to R. padi and (b) that genes putatively related to resistance towards a monocot specialist aphid, R. padi in the
monocot barley might confer resistance against a generalist aphid,
M. persicae in a dicot plant, Arabidopsis.
With regard to hypothesis (a), it was first considered whether there is any
previous evidence of a gene being up-regulated in response to an aphid
shown later to be conferring resistance against this aphid. Indeed, in
Arabidopsis, genes that were up-regulated in response to M. persicae
encoding for phytoalexin deficient 4 (PAD4) and a gene involved in
trehalose metabolism (TPS11) were shown to provide antibiosis and
antixenosis towards M. persicae (Louis and Shah 2015). PAD4 knockout
mutants and TPS11 overexpression resulted in higher and lower aphid population growth in comparison to the wild type Arabidopsis, respectively (Louis and Shah 2015). As a third example, silencing in tomato of an
α-dioxygenase that contributes to oxylipin synthesis and that was upregulated by M. euphorbiae enhanced M. euphorbiae population growth
(Avila et al. 2013).
Regarding hypothesis (b), first it will be considered whether one gene
might confer resistance against different (preferably both specialist and
generalist) aphids, secondly Arabidopsis-M. persicae as a model system in
plant-aphid research will be discussed and thirdly, it will be considered
whether there are examples of transfer of resistance traits from barley to
Arabidopsis.
There is more evidence that a certain gene is active against a specific
aphid species, than the opposite, possibly because genes that have been
studied are classical R-genes, involved in specific interactions. However, the
gene Mi-1 has a very broad organism spectrum and confers resistance
against certain nematodes, whiteflies, psyllids and aphids (Kaloshian 2004).
Notably, the resistance is specific for certain biotypes of M. euphorbiae and
it is not active against the generalist M. persicae. Furthermore, resistance
against specialist spotted alfalfa aphid (Theriophis trifolii Monell) and
specialist bluegreen aphid (Acyrthosiphon kondoi Shinji), in barrel medic
(Medicago truncatula Gaertn) cultivars were shown to be controlled by
separate genes (Klingler et al. 2007). One example of a resistance source
active against two different aphids is the case of D. noxia resistant barley,
27
which was shown to reduce the growth of both D. noxia and R. padi, but it is
not known if the resistance to the different aphids is controlled by the same
gene or not (Jimoh et al. 2011).
Arabidopsis thaliana (L.) is a useful model organism to study plant
processes because of its small genome size, short generation time, selfpollination and easy transformation process (Koornneef and Meinke 2010).
M. persicae is a cosmopolitan, polyphagous herbivore. M. persicae primary
(winter) host are trees of the genus Prunus, mainly the peach tree (Prunus
persica Batsch) and the aphid can use plants in more than 40 different plant
families as its secondary host. The secondary hosts include both dicots (e.g.
Brassicaceae, Cucurbitaceae and Fabaceae) and monocots (i.g. Poaceae,
Liliaceae and Araceae). The eggs overwinter on the primary host and in the
spring the egg hatches. The aphid has several generations on the winter host
before it migrates to the secondary hosts. In autumn, adult aphids migrate to
Prunus species, male and female mate and the eggs overwinter there.
M. persicae populations can be very dense on the plant and cause water
stress, wilting and reduce the plant growth and heavy aphid infestation can
reduce the yield of foliage and root crops (Capinera 2008). Arabidopsis and
M. persicae have a compatible interaction. The Arabidopsis genome is
sequenced and preliminary data from the genomic sequence of M. persicae
are available. In addition, several gene expression profiling studies of their
interaction have been done, as well as studies of M. persicae performance on
many Arabidopsis mutants. All this makes Arabidopsis-M. persicae a good
model system to study the genes and mechanisms involved in plant response
to aphids (AphidBase 2009; Moran et al. 2002; Louis and Shah 2013). In this
study, transformation of Arabidopsis made it possible to generate transformants in a relatively short time and to test the effects of our putative
aphid resistance sequences.
Whereas Arabidopsis is a good model plant for studying dicots, purple
false brome (Brachypodium distachyon (L.) Beauv) is now emerging as a
model system for studying monocots. There are several reasons, including
small stature, self-pollination, short life cycle, small genome and close
relation to the important crops wheat and barley. Several molecular
resources have been developed for this plant such as germplasm collections,
genetic markers, a genetic linkage map, bacterial artificial chromosome
libraries, physical maps, mutant collections, microarrays and databases as
well as highly efficient transformation protocols (Vogel et al. 2010).
B. distachyon has good potential as a model in studying plant-pathogen/pest
interactions (Fursova et al. 2012; Sandoya and de Oliveira Buanafina 2014).
For example it was shown that greenbug did not survive on Arabidopsis and
performed poorly on rice whereas it performed well and responded to
B. distachyon similarly to what was observed in wheat (Azhaguvel et al.
2009). The evaluation of Russion wheat aphid symptoms on B. distachyon
accessions showed varied levels of resistance to this aphid, enabling search
28
for QTLs and genes related to this interaction (Sandoya and de Oliveira Buanafina 2014). Similar studies of R. padi performance on B. distachyon are
still waiting to be carried out.
There are several studies where a gene from a monocot was expressed in
Arabidopsis and resulted in a phenotypic change. For example, in two separate studies, Arabidopsis with genes responsible for abiotic stresses in wheat
(salt stress and hyperosmotic stress) were reported to boost root growth
under salt stress and significantly raise tolerance to salt, drought and freezing
stress as well as root system improvement under hyperosmotic stress (Ge et
al. 2007; Tian et al. 2013). In addition, a gene responsible for heat stress
from barley increased thermotolerance when overexpressed in Arabidopsis
(Shi et al. 2001).
In this paper, the cDNA encoding a proteinase inhibitor (PI) in barley was
cloned and transformed into Arabidopsis. PIs are small proteins. They have a
role in plant defense against herbivores and pathogens and some are known
as storage proteins (Fan and Wu 2005). PIs are present in seeds and their
production is induced in other tissue in response to herbivore attack or
wounding (Koiwa et al. 1997). They inhibit digestive enzymes in insect
midguts and decrease the essential amino acids for insect growth and development (De Leo et al. 2002). PIs are ubiquitous proteins in plants (Fan and
Wu 2005). The first time the possible role of PIs in plant defense was reported was by showing that the larva of red flour beetle (Tribolium castaneum
Herbst) could not develop normally on soybean flour and grits (Mickel and
Standish 1947). The defensive roles of PIs have therefore been studied in
many plants. Rearing on artificial diets containing PI caused growth inhibition of M. persicae and expressing a gene encoding the same PI in oilseed
rape decreased M. persicae weight and fecundity (Rahbé et al. 2003). In
barley leaves, the levels of two different PIs increased twofold upon R. padi
infestation in an aphid resistant cultivar and feeding on artificial diet containing the purified PIs decreased R. padi survival (Casaretto and Corcuera
1998).
Aphid behavior and performance on transgenic Arabidopsis and barley
with a putative aphid resistance sequence from barley (Paper IV)
In this paper a barley sequence representing an open reading frame (ORF)
encoding a protein with similarity to a part of a kinase was isolated and
transformed into Arabidopsis and barley and the interaction with M. persicae
and R. padi were evaluated, respectively.
Plants sense changes in the environment including those causing biotic or
abiotic stress. The information from outside of the cell is mediated intracellulary by signal-transducing mechanisms and protein kinases are involved
in such transductions (Champion et al. 2004). Protein kinases transfer
29
phosphoryl groups from ATP to amino acid residues of a protein. Based on
their primary sequences and the amino acid residues phosphorylated they are
classified into three groups; serine/threonine kinases, tyrosine kinases and
histidine kinases (Chevalier and Walker 2005). Some of the known plant
resistance genes encode a serine/threonine kinase and confer resistance to
pathogens, for example Pto that confers resistance to Pseudomonas syringae
pv. tomato (Martin et al. 1993), Rpg1 that confers resistance to Puccinia
graminis f. sp. tritici (Brueggeman et al. 2006) and Stpk-V that confers
resistance to Blumeria graminis f. sp. tritici (Cao et al. 2011).
Receptor-like serine/threonine kinases play a major role in a variety of
processes from growth and development to response to pathogens. They are
a family of transmembrane protein with similar structure (Afzal et al. 2008).
In Arabidopsis, the ERECTA gene, known to be encoding a receptor-like
serine/threonine-protein kinase, conferred resistance against bacterial wilt
(Ralstonia solanacearum Smith) (Godiard et al. 2003). Another example is
the BAK1 gene that encodes a receptor-like serine/threonine-protein kinase
involved in resistance against Pseudomonas syringae (Van Hall) and
Hyaloperonospora arabidopsidis (Gäum) (Roux et al. 2011). This gene was
also found to be involved in resistance against M. persicae in Arabidopsis
(Prince et al. 2014). A transcriptomics study on cucumber identified several
genes encoding receptor-like serine/threonine-protein kinases as defense
factors against A. gossypii (Liang et al. 2015).
30
Main results and discussion
In paper I, the purpose was to investigate the possible relation between the
expression of certain genes and aphid resistance characteristics in a selection
of barley breeding lines consisting of 23 genotypes. The genes were selected
based on the results of a comparative study between aphid resistant and
susceptible genotypes after R. padi infestation (Delp et al. 2009). Out of nine
investigated genes, five were expressed as we predicted. Two gene
sequences (thionin (THIO) 1570 and PI) had higher constitutive transcript
abundance in resistant compared to in susceptible genotypes. Two of the
sequences (AOS and NMT) were expressed equally in all the genotypes and
lipoxygenase (LOX)2 was expressed higher in susceptible genotypes. Three
other genes were either not expressed in some of the genotypes (β-1,3glucanase III (ßGLU-GIII) and the sequence corresponding to
contig16360_at (STK)) or were not expressed in any of the genotypes (barley
chemically induced (BCI)-4). Based on the results from paper I, we cannot
definitely assign any of the investigated gene sequences as susceptibility or
resistance factors towards R. padi, but the study gave support to the putative
role of THIO1570, LOX2 and PI as significant in the interaction between
barley and R. padi (Fig. 2 and Table 3 in paper I).
In paper II, the role of three ß-1,3-glucanases in aphid susceptibility was
investigated in 15 barley breeding lines with known R. padi resistance
characteristics based on aphid growth. A time course study of aphid settling
was carried out and the transcript abundance of three ß-1,3-glucanase
sequences (contig1636_at, contig1639_at and contig1637_s_at) were examined on the same plant leaf tissue. The previous comparative study on two
resistant and two susceptible barley genotypes suggested a susceptibility role
for sequences corresponding to contig1636_at and contig1639_at, but
showed no difference in the transcript abundance of the sequence corresponding to contig1637_s_at between resistant and susceptible genotypes
(Delp et al. 2009). In this study of 15 barley genotypes, ten genotypes
expressed contig1636_at (Fig. 2 in paper II). The contig1639_at sequence
was induced in 13 genotypes and the contig1637_s_at sequence in 7 genotypes upon R. padi infestation (Table 3 in paper II). A comparison between
aphid settling and former resistance characterisations of the barley lines
based on nymphal growth revealed that in all cases with significant differences in settling, the susceptible genotypes had higher aphid settling than
resistant genotypes. Furthermore no resistant genotype had higher transcript
31
abundance than susceptible genotypes when fed on by R. padi. This
suggested that certain ß-1,3-glucanases might be susceptibility factors in the
R. padi-barley interaction.
In paper III, the possible effect of a barley proteinase inhibitor gene (BCI7) expected to have a function in R. padi resistance (Delp et al. 2009) was
investigated by transforming the cDNA into Arabidopsis. The effect was
evaluated by studying the behaviour and growth of M. persicae. The
Arabidopsis plants were transformed with either a constitutive or a phloem
specific (ps) promoter. The evaluation showed that the number of aphids was
lower on BCI-7 and psBCI-7 transformants compared to on control plants
after 5 days. In addition, a significant difference was observed between one
BCI-7 transgenic line and controls in choice tests. These results indicated
that the expression of BCI-7 in Arabidopsis increased aphid resistance.
In paper IV, the effect of an ORF from barley, with 55 % similarity to a
part of an Arabidopsis putative serine/threonine kinase sequence that was
found up-regulated by aphids in resistant barley (Delp et al. 2009) was investigated. The sequence was transformed into Arabidopsis and barley and the
effect of transformation on aphid performance was analyzed. There were no
differences in M. persicae population growth assessments and in choice tests
between transformed Arabidopsis and control plants (Fig. 2 and Fig. 3 in
paper IV). The barley plants were transformed with a constitutive and a
phloem specific promoter, resulting in selected lines named p:5, p:8 and
ps:1, respectively. There was no relation between the transcript abundance of
the transformed sequence and aphid numbers in a population growth test
(Fig. 4 in paper IV). Taken together the results suggest that there was no
effect of the transformation and that this might be due to that part of the ORF
cloned does not contain domains important in resistance against stresses
(Suppl. Fig. 1 in paper IV).
32
General discussion about methods
Still based on the idea that the analysis of the transcript abundance of certain
genes may give indications of differences between genotypes, there are other
possible approaches than the one applied here. Our selection of candidate
genes was based on a microarray study (Delp et al 2009), which is a method
that gives a broad overview of gene expressions. At the time when
the microarray study was carried out, a barley chip was available that only
contained about half of the barley genome, thus the study could have missed
a number of interesting genes. Nowadays, it is becoming increasingly common to use the RNA sequencing method (RNA-seq) for the quantification of
transcriptomes. This method has clear advantages over using the microarray
method. Firstly, it does not depend on prior genomic sequence knowledge,
which is advantageous for non-model organisms. Secondly, it has a larger
dynamic range compared to the microarray method. Very high and very low
transcript levels are detectable as well as all the isoforms of a gene. This is a
significant advantage compared to the microarray method where a probe
might hit just some of the isoforms of a gene and as a result the probe does
not show the expression of the whole gene complex. However there are
some limitations in RNA-seq as well, e.g. that data analysis is complex,
expensive and needs special computers and generate lots of data which is
difficult to share and costly to store (Zhao et al. 2014).
Other approaches to study overall differences between genotypes could
be to study proteomics and metabolomics. Proteomics is a method to study
structure and function of all expressed proteins and provides information
about proteins actually being present and possibly interacting with a
pathogen or herbivore, which is its advantage over transcriptomics. The disadvantage is that it´s protocol is not as straightforward as transcriptomics
and the data analysis is complex (Chandramouli and Qian 2009).
Metabolomics is a method to identify and quantify metabolites. It is a
sensitive and rapid method compared to transcriptomics and proteomics,
needs little sample preparation and generates a lower number of endogenous
molecules relative to the numbers that are analyzed with transcriptomics and
proteomics. However, due to the diverse chemical structures with different
abundance, there is not a good general method to study all the metabolites.
The method needs complex bioinformatic analysis or analytical platforms
and is expensive (Roessner and Bowne 2009).
33
The major reason for our choice of approach was that some candidate
sequences for R. padi resistance or susceptibility in barley were already
identified in the microarray study (Delp et al. 2009) (paper I and II). We
aimed to do functional genomic studies by cloning and expression of the
candidate genes Arabidopsis and barley (paper III and IV).
In paper I, the transcript abundance of susceptibility- and resistancerelated candidate genes were analyzed in a selection of barley breeding lines
with different levels of resistance and in paper II, the constitutive and
induced transcript abundance of two susceptibility related candidate genes
and one additional gene (altogether three ß-1,3-glucanase sequences) were
investigated in a selection of barley breeding lines with different levels of
resistance.
To analyze the transcript abundance of the selected genes, RNA was
extracted from frozen leaf tissue and was reverse transcribed to synthesize
cDNA and then the transcript levels were assessed using quantitative polymerase chain reaction (qPCR).
A big sample size will increase precision in the statistical analysis
(McDonald 2009). In this study, because of logistic reasons the sample size
was 3 biological replicates, each consisting of one individual plant. This
weakness is somewhat balanced by the usage of a large number of genotypes, which had been characterized with regard to aphid antibiosis.
In papers III and IV, two candidate sequences for R. padi resistance
identified in the microarray study (Delp et al. 2009) were cloned and transformed to Arabidopsis ecotype Columbia (Col-0) and barley cultivar Golden
Promise. Gene silencing and gene addition (or overexpression) are two of
several approaches that are used in functional genomics to study the function
of a certain gene sequence. It may also be used in applications e.g. to
improve crop resistance against pathogens and insects. Gene silencing technology is referring to down-regulation or interruption of the expression of a
specific gene. Gene silencing in monocots could be obtained by several
methods including RNA-mediated interference (RNAi) (Travella et al.
2006), virus induced gene silencing (VIGS) (Anderson et al. 2014), antisense
oligodeoxynucleotides (ODN) (Xie et al. 2014) and clustered regularly
interspaced short palindromic repeats (CRISPR)/Cas9 (Jiang et al. 2013).
All four methods are rapid and inexpensive. On the negative side, it may be
noted that they may result in off-target effects and that they are not developed for all plant species. RNAi, VIGS and ODN methods result in partial
silencing with variable levels and complete silencing is rare (Gilchrist and
Haughn 2010; Dinç et al. 2011).
Using the RNAi method it is possible to knock down multiple
homologous genes, in a tissue specific or temporal manner, but some genes
are not possible to silence with this method and the method needs transformation (Gilchrist and Haughn 2010). VIGS was used widely in the past few
years for characterizing the function of plant genes (Gupta et al. 2013). It is a
34
rapid method and the results are obtained in 3 to 4 weeks from infection to
gene silencing with transient inhibitory effect and unlike the RNAi method
does not need transformation (Burch-Smith et al. 2004). The application of
this method in plant resistance against fungi, bacteria, nematode and
herbivores including aphids were reported (Huang et al. 2012). Negative in
this context may be that virus sequences induce defense responses (Unver
and Budak 2009).
There are some functional genomic studies on plant-aphid interactions
using the silencing methods described above; for example silencing a
resistance and a susceptibility gene in wheat by means of VIGS in two separate studies characterized the function of these genes against D. noxia (van
Eck et al. 2010; Anderson et al. 2014).
The ODN method is a widely used method in animal research and it has
been shown to be an efficient method for gene silencing in plants including
wheat and barley. In this method, a single-stranded ODN enters the cell,
binds to its complementary RNA and inhibits gene expression (Dinç et al.
2011; Xie et al. 2014). It has as advantages over VIGS and RNAi that ODN
design and synthesis are very fast and simple and that the inhibitory effect
can be obtained within 24 hours. On the negative side, the inhibitory effect is
transient, and the plant tissue needs to be cut and transferred to the solution
containing the ODN, thus the plant is not intact which is desirable in studies
of plant-aphid interactions (Sun et al. 2005; Dinç et al. 2011).
CRISPR/Cas9 has recently developed as a powerful genome editing tool
and can be used to functionally inactivate target genes. In this method a
single strand RNA enters the cell and makes a complex with Cas9 protein.
The complex binds to complementary DNA and cuts it. This method has
been successfully applied to various plant species including N. benthamiana,
A. thaliana, rice, wheat and barley (Shan et al. 2013; Lowder et al. 2015;
Lawrenson et al. 2015). Successful CRISPR/Cas9 knocks out the target
genes whereas other methods mentioned above mediate gene knock down.
This method requires clone isolation that makes it time consuming compared
to other methods (Lawrenson et al. 2015).
Another approach is gene addition or overexpression. There are several
methods to introduce DNA into host plant cells. Direct techniques are based
on cell wall penetration physically (e.g. bombardment, electrophoresis,
vacuum infiltration) or penetration into protoplasts using e.g. ethylene glycol
or calcium phosphate. Indirect techniques are based on biological methods
where a plasmid construct is introduced into the target cell by means of
microorganisms, e.g. bacteria or viruses (Chung et al. 2006; Rao et al.
2009).
Agrobacterium tumefaciens is a gram-negative bacterium found in soil. It
causes crown gall disease in plants and is widely used as a tool to develop
stable transformed plants. A. tumefaciens has a tumor-inducing (Ti) plasmid
and is able to transfer and integrate a part of the Ti plasmid called T-DNA
35
into the host plant. A. tumefaciens can deliver a desirable sequence with
T-DNA to a broad range of plant species (Gordon and Christie 2014).
Agrobacterium-mediated plant transformation is the most common method to transform Arabidopsis and barley (Bent 2006; Harwood 2014).
Arabidopsis transformation using floral dip method is simple, reliable, with
low cost and no needs for tissue culturing that may cause somaclonal variation (Bent 2006). Barley transformation using the immature embryo method
is highly efficient and produces transformants with low copy numbers of the
transgene, stable transgene expression and rare transgene silencing (Bartlett
et al. 2008).
36
Conclusions
Much effort has been used to develop cultivars resistant against aphids.
There are barley cultivars bred for resistance against S. graminum and
D. noxia (Porter et al. 1998; Smith and Chuang 2014). This resistance has
been selected based on screening for symptoms and the molecular basis of
the resistance is not identified (Mornhinweg et al. 2009). No barley cultivar
with resistance against R. padi is available.
Identifying genes related to susceptibility or resistance against aphids is
important to explain plant-aphid interactions and for use in resistance breeding. This thesis contributes to such knowledge with regard to R. padi in
barley and the results can be summarized as:
•
Genes coding for a thionin and a PI were identified as putatively related to resistance and a gene for a lipoxygenase as putatively related
to susceptibility against R. padi.
•
Two ß-1,3-glucanase sequences were identified as possible susceptibility factors against R. padi.
•
The cDNA encoding the PI from barley mentioned above caused
increased resistance against M. persicae when expressed in
Arabidopsis.
•
A barley sequence earlier shown to be up-regulated in response to
R. padi in moderately aphid resistant genotypes and with unknown
function, did not increase resistance against M. persicae in
Arabidopsis nor against R. padi in barley upon transformation.
37
Suggestions for further studies
The transcript abundance of the constitutively expressed candidate gene
sequences for aphid resistance or susceptibility identified in paper I could be
studied in a time course study with and without aphid (i.e. the plant material
used in paper II).
The functional role of the candidate genes from papers I (thionin, proteinase inhibitor (PI=BCI-7) and lipoxygenase) and II (ß-glucanases;
contig1639_at and contig1637_s_at) could be analyzed by gene silencing
and/or overexpression in barley followed by evaluation of R. padi growth
and behavior. Transformation of barley for the overexpression of BCI-7 has
already been carried out and the evaluation has started. Besides the study of
R. padi, it would be interesting to study M. persicae on transgenic barley to
find out the effects on this generalist aphid.
B. distachyon is a good model plant which can be used instead of barley
to do a functional study on the candidate genes. To do that, first it is necessary to study R. padi performance on B. distachyon.
Studies of the tissue localization of the proteins encoded by the candidate
genes would add to the understanding of their functions.
To study the direct effect of the candidate sequences on aphid development and behaviour, the cDNAs can be expressed in Escherichia coli, the
protein can be purified and the purified protein can then be used in artificial
diets. This approach is suitable to examine the effect of the proteinase inhibitor (PI=BCI-7).
As ultimate tests, transgenic barley lines overexpressing or silenced for
above candidate genes should be evaluated in field trials with natural or artificial aphid infestations.
38
Acknowledgements
I sincerely thank everyone who has been helpful in my studies.
To start I would like to thank my supervisors Dr. Lisbeth Jonsson, Dr. Inger
Åhman and Dr. Lisa Beste for their kind support during these years.
I am also grateful to Dr. Katharina Pawlowski for her advice, incredible
kindness and friendship.
Many thanks to my office-mate Dr. Afaf Hamada and my colleagues
Aleksandra, Andrea, Lotta, Marco, Sandra, Sofie, Van and last but not the
least Denis, who always was there to answer my questions.
I also want to acknowledge all the present and past members of the Plant
Physiology division.
Many thanks to Sara, Behnaz, Afshin, Bahareh and special thanks to Farzad
for their friendship, support and kindness. Finally a warm thanks to my
beloved parents and my siblings for their love, endless support and encouragement.
This work got financial support from the Swedish Foundation for Strategic
Environmental Research (Mistra) via the PlantComMistra program and from
CF Lundström Foundation.
I also gratefully acknowledge support in the form of stipends from The Royal Physiographic Society in Lund (the Nilsson Ehle - donations), Helge
Ax:son Johnson Foundation and Stockholm University donation funds as
well as travel grants from the Department of Ecology, Environment and
Plant Sciences.
39
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