1Plant microbe symbioses allow
nutrient and sugar exchange
2Ectomycorrhizal Symbioses
fungi colonize only the apoplastic space between plant cells
3Endomycorrhizal symbioses
fungi colonize the space within plant cells; also includes AMF
4arbuscules
highly branched structures that fungi and plant form for nutrient and sugar exchange within the root cortex cell layer
5SLs were isolated as
branching factors secreted by the plant and received by AMF
6the bioactive form of strigolactone is generated through a pathway involving the enzyme
ORT1
7how was plant branching signal identified?
purify root exudates, size fractioning, branching assays, HPLC
8how was myc factor identified?
isolated exudants of mycorrhized carrot roots, fractioned with solvents, assay on GUS reporter system, Look for fractions that give positive GUS response
9Myc factor is actually _____ and _______
LCOs; COs
10LCOs
trigger a variety of symbiosis phenotypes in plants, and are also involved in interactions with rhizobia
11COs
trigger AMF formation in both legumes and non-legumes at even low concentrations; Considered universal pre-symbiotic AM signals
12AMF colonization takes place through a coordinated process involving the plant’s production of a growth space, called the
pre-penetration apparatus (PPA)
13SYMRK and CERK1 complex sense ______ and interact with _______ to produce _______ and ______
COs; HMGR1; mevalonate; signal B
14signal B travels through ______ and creates _________ in the nucleus
NUPs; Ca+ oscillations
15Ca+ oscillations are controlled and compensated for by the action of _______ (Ca+) and _________ (K+)
MCA8 and CASTOR/POLLUX
16Ca+ spikes are sensed by ______ which phosphorylates downstream targets and turns on _______
CCaMK; AM machinery
17Once Arbuscules form ______ and _____ allow exchange of sugars
STR; STR2
18MtPT4
AMF-specific phosphate transporter MtPT4
19periarbuscular space (PAS)
space between cell wall and fungal cell wall where exchanges take place
20how to create RNAi
create DNA construct containing an inverted repeat that will match our target mRNA (the one we want degraded), and introduce to WT with T-DNA mutagenesis. when transcribed it will create a hairpin loop fragment in the RNA and a section of double stranded RNA
21siRNAs are recognized by ______ which uses them to hunt down additional ______ within the cell that matches the siRNA sequence
AGO1; mRNA
22AGO1 will _____ mRNA, and ____ will turn the product into additional dsRNA copies, creating a feed-forward loop.
cleave and destroy; RDR6
23dsRNA is recognized by _____in the cell and ____
Dicer proteins (DCL); chopped up into small siRNA fragments
24in the MtPT4 RNAi mutant, ______ accumulates, indicating _______
Polyphosphate; inability of the plant to accept its transfer from the fungi
25decreased Pi exchange is linked to
aborted symbioses
26Common Symbioses Pathway (CSP)
a set of core genes is involved in the establishment of both AMF and rhizobia-legume relationships
27Rhizobium Legume Symbioses (RLS) are restricted to
a subset of eudicots (Fabacea)
28RLS research is easier than AMF because
ease of culturing rhizobia, their amenable genetics, and the advantages of legumes as model plants
29 no eukaryotic enzymes have been discovered which can
break the triple bond of N2
30reduction of N2 to NH3 is catalyzed by
nitrogenase in prokaryotes
31in RLS, the purpose of the nodule is
increase nitrogen availaiblity for the plants, in exchange for carbon for the rhizobia
32The RED color within the nodule is
leghaemoglobin, which reduces local O2, since nitrogen fixation is oxygen sensitive
33Mutants deficient in RLS identified through
EMS
34DMI genes
does not make infections (when mutated)
35GUS was used to study RLS and identified 3 mutants that could not
could not form nodules and could not induce ENOD11 expression
36Calcium spiking is lost in ______ knockouts, but not _____. therefore ______
dmi1 and dmi2; dmi3; DMI1 and DMI2 were placed upstream of Ca+2 spiking, while DMI3 was placed downstream
37The N terminal domain of DMI1 is similar to
a potassium channel from a Methanobacterium
38The C-terminus of DMI1 contains an RCK domain, which is
calcium-regulated gating ring that controls the activity of the potassium channel
39early work to decode calcium relied on
dmi mutants and injection of calcium sensitive dyes
40new tech for decoding calcium spikes
fret based sensor (calmodulin)
41Calmodulin
small protein with 4 binding domains; when bound to calcium it becomes active and passes energy to the YFP domain
42FRET allowed us to determine
Ca2+ spiking is cell-type specific, localized to the nucleus
43Nod factor-induced nuclear calcium oscillations originate from
periphery of the nucleus, where the maximal changes of calcium occur, further suggesting that calcium enters the nucleus from the inner nuclear envelope
44in DMI3, the kinase promotes
nodule formation
45in DMI3, EF hands and Autophosphorylation of T271A ______
46in DMI3 ______ is required for nodule ihibition
CaM
47Low levels of Ca2+ binding to the EF-hands of DMI3 promotes ___________ and inhibits _______
autophosphorylation; kinase activity
48High levels of Ca2+ promotes _____, opening up the protein and relieving autoinhibition, thus activating ________
CaM binding; the kinase activity
49DMI3 that only responds to
high spikes of Ca2+
50steps of rhizobia detection
COs detected by SYMRK, which phsophorylates HMGR1, who sends a signal to nuclear calcium channel, leading to a burst of calcium in the nucleus sensed by CCaMK. calcium spiking activates castor/pollux which sends out K+
51During the infection of nodule cells, rhizobia differentiate into
bacteroids
52bacteroids surrounded by plant membrane are called
symbiosomes
53Expression of _______ in the symbiosomes allows for a low O2 environment that can still respire rapidly
laghaemoglobins
54to control the growth of the rhizobia within the plant tissue, the symbioses involves a complete inactivation of
glutamine synthase
55all nitrogen fixed by the rhizobia must go ______ and rhizobia must receive their nitrogen from the plant in _____
to the plant ; an alternate form (amino acids)
56Carbon (in the form of ______) and _______are supplied to the mature
_______ in exchange for NH4+
malate; amino acids; bacteroids
57AMF vs RLS calcium spikes
AMF more chaotic; RLS have long down phases; hair and non-hair proteins different
58Primary root forms during
embryogenesis
59Lateral roots form mostly
after germination
60At the globular stage, the hypophysis divides asymmetrically to form
precursors of the _______ and the ______
Quiescent Center (QC); columella root cap
61QC is _____ located in ______
4 to 7 cells; RAM
62QC divides _____ and promotes
slowly; division of initial cells that produce the cell files surrounding QC
63auxin accumulation organizes the RAM by
orienting the QC cells at the maxima
64PLT expression domains overlap at the
QC and in initial cells
65____ activity required to sustain PLT expression in the RAM
MP
66High combined PLT levels promote
stem cell activity in RAM
67Moderate PLT levels promote
division of stem cell daughters
68Low PLT levels allow
cells to differentiate
69PLT genes encode
AP2 domain transcription factord
70PLT mis-expression in the shoot region of a developing seedling causes
root formation
71cortex and endodermis cells, collectively called
ground tissue
72hort-root (shr) and scarecrow (scr) mutants only form
one layer of ground tissue: cortex
73SHR and SCR required for
periclinal division of the cortical/endodermal initial (CEI) cell
74SHR and SCR encode
GRAS domain transcription factors
75SHR mRNA is expressed only in
stele cells
76SHR protein moves from
stele to CEI cell
77SHR protein directly induces
SCR expression in CEI
78SCR forming a complex with SHR prevents _____ and induces _____ to promote CEI division
he movement
of SHR into outer cell layers of the root; cell cycle genes
79PLT and SCR (and TCP) proteins
physically associate into complex
80PLT-SCR protein complex binds to and is necessary to express
WOX5 promoter in QC cells
81WOX5 is required to
prevent the differentiation of the adjacent columella stem cells
82columella stem cells divide
downward to form root cap
83CLE40 is expressed in ____ and regulates _____
distal columella cells of the root cap; WOX5 expression
84CLV1 and ACR4 bind to ____ and form a complex, expressed in _______
CLE40; columella stem cells
85CLE40 induces ____ but not ___
ACR4; CLV1
86clv1 mutant roots contain
excess columella stem cells
87signaling by CLE40 through the CLV1-ACR4 complex limits
number of columells stem cells
88CLE40 signaling via CLV1-ACR4 represses
WOX5 expression
89WOX5 directly binds to _______ to repress expression; thus maintaining
CDF4; columella stem cell fate
90how will stomate respond to climate change
decreased stomatal density over time
91Herbaria are rich data sources to study:
global plant diversity, plant domestication, and plant responses to global change
92Herbaria can track and preserve
morphology; DNA, biotic interactions and isotopes and elements
93DNA from herbaria is
fragmented, metagenomic and damaged
94what must plants defend against
viruses, microbes, nematodes, herbivores
95major entry points for pathogens
stomate, wounds, intracellular space, cell wall degredation, insect delivery
96Plants respond to necrotrophic pathogens by inducing defense systems that generate _____ and try to _____
antimicrobial compounds; limit cell death
97Plants often respond to biotrophic pathogens infection by
killing surrounding cells
982 defense mechanisms
physical and chemical barriers;; sensing machinery to detect pathogen presence
99before invasion triggers ____; after invasion triggers ______
external sensors activate PTI; internal sensors activate ETI
100PTI is different than ETI by
before invasion; conserved proteins; slower; effective against non-evolved
101ETI is different than PTI by
after invasion, specific proteins; effective against adapted pathogens; stronger and quicker
102PAMPS are recognized at ______ by proteins that bind specific microbial
PAMPS and trigger internal ______
the plasma membrane; PTI
1032 PAMP examples
bacterial flagellin; fungal chitin
104when flagellin is recognized
FLS2-BAK1 complex sends an intracellular signal into the cytoplasm of the host cell and phosphorylation cascade
105PTI in plants is similar to ______ in animals, but _____
innate immunity; convergent evolution
106Type-III secretion system (TTSS).
needle system that secretes bacterial effectors into host cell
107In contrast to PAMPS, effectors are
variable and dispensible to pathogen survival
108Bacteria typically use ____, Fungi and Oomycetes use ______, and Nematodes use _____
TTSS; haustorium; stylets
109effectors are recognized by these 3 models:
direct, guard, bait
110direct model
effector is sensed through direct binding to the NB-LRR
111guard model
effector binds its host target which induces a confirmational change in the host target that is sensed through direct binding to the NB-LRRprotein
112bait model
effector binds a host bait protein and is then sensed through direct binding to the NB-LRR; the host bait has no other host function
113PopP2 is
effector used to dampen PAMP response
114PopP2 binds to and inactivates
WRKY transcription factors that activate PTI
115RRS1-R is an _____ that interacts with ____ through direct binding. Binding leads to downstream ________
NBLRR
protein; PopP2; activation of ETI responses
116_____ is an essential part of the plant’s PTI recognition of the bacterial pathogen Pseudomonas
RIN4
117Pseudomonas infect cells with two effectors ______ and _______, which act to inactivate ____ through phosphorylation or degradation
AvrRpm1; AvrRpt2; RIN4
118The NB-LRR proteins ______ and ______ act to sense modified or degraded RIN4, and turn on ETI defenses
RPM1; RPS2
119which model is harder for pathogens to evolve against
guard
120BAK1-FLS2 induces ______ and reduces ______
immunity; growth
121BAK1-BRI1 stimulates ______ and represses
growth; immunity
122in old leaves, it has been observed that _______, which
prioritizes the response to _______
ABA represses SA synthesis; abiotic stress over biotic
123at another interactor, ______, represses ______-, which prioritizes the response to _____
PBS3; the ABA-induced repression of SA synthesis; biotic over abiotic stress
124Arabidopsis aos mutants are
JA deficient
125OPR3-OE plants make 30% more
JA
126a pathogen is detected by ETI or PTI and induces SA via _______ pathway
shikimate biosynthesis
127SA triggers and reinforces a ______ which monomerizes NPR1, allowing it to ______
redox shift; enter thre nucleus
128When levels of SA are low NPR1
is bound to NPR4 and led to death
129When levels of SA are too high, NPR1 is
bound to NPR3 and also led to death
130at right levels of SA, NPR1 induces defense genes ______ and _______
TGA; PR1
131dispersal of MeSA allows for
SAR
132Necrotrophic effectors activate ________ in order to ________
the SA pathway ; downregulate the JApathway.
133Biotrophic effectors activate ______ to ________
the JA pathway; suppress the SA pathway
134coronatin is a _______ that stimulates
JA-Ile mimic; JAZ ubiquitination
135HopZ1A effector _______, which leads to their degradation
acetylates JAZ
136AvrB effector ______, which triggers _________ through _____
phosphorylates MPK4; JA signalling; RIN4
137HopX1 effector induces ______ by ________
JAZ inactivation; directly degrading them
138Beneficial fungi produce a chitin-binding effector ______ that prevents activation of plant immunestimulation
Ecp6
139Nitrogen fixing bacteria use an extracellular protease, _____, that degrades theflagellin MAMP
APRA
140Rhizobia-produced Nod factors interact with a receptor _____ to reduce levels of __________, preventing detection. of flagellin and induction of PTI
Lyk3; FLS2 and BAK1
141Rhizobia also produce ______, extracellular sugar matrices that can prevent _______
LPS and EPS; Ca2+ and ROS signaling
142byprodoct of oxidative phosphorylation in the mitochondria
ROS
143excess ROS leads to
membrane damage, protein oxidation, DNA damage, and programmed cell death
144Hydroxyl radicals react with membrane lipids through
lipid peroxidation, producing reactive aldehydes and membrane leakage
145Alternative Oxidase (AOX) is _____
a plant mitochodrial specific solution to conditions of high ROS production
146ROS scavengers are
enzymes whose function is to convert ROS back to water
147Rbohs are
a family of eukaryotic protein that function to produce short bursts of ROS in the apoplastic compartment in response to a cytoplasmic signal
148Rboh function is mediated by
EF hand domains that bind Ca+
149activation of Rboh involves ______ that activates ______
Ca+ stimulating the EF hands; phosphorylation serine residues
150The Ca2+ channel blocker lanthanum inhibits _______ and blocks ______
Ca+ transmission; ROS-regulated transcriptional markers, such as ZAT12
151ROS and Ca+ cycle
ROS and Rboh produced; ROS perceived by neighbore and induces Ca+ spike, which activates CPK5, which phosphorylates and activates Rboh, which produces ROS
152why is ROS wave cycle in the apoplast?
less ROS scavengers; doesn't hurt cellular function
153calcium wave happens primarily in
cytoplasm
154ABA induction in root leads to rapid induction of ____, which eventually causes ______ by activating ______
RbohF; stomatal closure; SLAC1
155stomatal closure involves
rapid movement of potassium and chloride ions out of the vacuole
156A slac1 mutant cannot ____ because it fails to ______
close stomata; transport K+ and Cl-
157abiotic stress induces _______, while biotic stress induces
RbohF; RbohD
158Waterlogging =
excess of water surrounding the root system
159waterlogging induces ______ with ______
RbohH; ethylene
160Aerenchyma =
air filled pockets in the root system that aid in gas exchange
161Root hair growth, is controlled in part by ______, which is expressed only in _____
RbohC; hair cells
162RHD6 expression by GL2 in non-hair cells promotes ____
RbohC
163rbohc mutants can _____ but fail to ________
initiate root hairs; elongate them
164cell wall lossening by RbohC allows ______ which leads to ______
turgor-driven polar cell expansion; root hair growth
165SAR requires
ROS, G3P, and pipecolic acid
166Skotomorphogenesis
upward development of seedlings in complete darkness
167Photomorphogenesis
development of photosynthesis machinery, and the slowing of upward growth in response to adequate light
168shade avoidance
upwards growth of the stem in response to a shift light wavelength due to another plant’s shade
169phytochromes sense light often with the help of
chromophore molecules
170each phytochrome has
a pocket within its protein structure that houses a bilin chromophore
171conformational shift in the phytochrome protein during exposure to light is driven by
light induced shift in bilin chromophore molecule
174phtyochromes are conserved by plants also have
extra PAS domains
175skotomorphogenesis produces
etiolated tissue responses including, longer+weaker hypocotyls and stems, increased internode length, and pale yellow color
176pifq mutant lacks
skotomorphogenesis
177what was used to identify what genes PIF3 binds to and activates
CHiP-Seq
178Loss of PIFs is accompanied by _____
transcriptioal changes in target gene PIL1
179A lack of light leads to PIF activity, which includes activation of ______, that leads to _______
PIL1; skotomorphogenesis
180active Pfr moves to the nucleus where it leads to _______
phosphorylation and ubiquitlaytion of the PIF protein
181Two other proteins _______ regulate parts of the shade avoidance pathway independently of the PIFs, but still relying on _____
SPA and COP1; phytochromes
182SPA and COP1 bind to and degrade the _____ , thus inducing
HY5 transcription factor; shade response
183in light, Pfr sequesters SPA and COP1, releasing ______ to ______
HY5; repress shade response
184Both PIFs and HY5 bind
G-box proteins
185Pfr/Pr also sense
temperature
186A central clock protein, _____ regulates _____
TOC1; PIFs
187PIL1 is ____ in the context of circadian rhythm
dawn-phased (turns on at dawn)
188TOC1 and PIFs antagonistically regulate
PIL1
189what method shows TOC1 interacts directly with PIF3
BiFC
190How does BiFC work?
Two proteins are each fused to half of a functioning YFP fluorescent marker; if the proteins interact, then YFP is reconstituted and signal is detected
191types of protein-protein interaction techniques
Co-IP, FRET, BiFC
192types of protein-DNA interaction techniques
DNAse footprinting, ChIP-Seq, Gel retardation assay
193DNA Detection method
DNA blotting (see T-DNA lec)
194RNA detection methods
RNA Seq, qPCR
195reporter assays and stains
GUS, Luciferase, GFP, Oregon green
196Describe the two things necessary to form an auxin maxima in the root tip
Auxin bulk flow via stele/vasculature from source leaves; Auxin flow up from root cap back up
197What would be the phenotype in the RAM if you made a 35S::CDF mutant? Why?
Differentiated CSCs, likely stunted root growth; CDF4 overactivity inhibits stem cell fate
198What morphological change would you expect to see in plants experiencing phosphate stress?
more root hairs; ETC1 would be turned on by WER under phosphate stress and inhibit GL2
199why wouldn’t a plant turn every epidermal cell into a hair cell?
more hairs might lead to too much of a nutrient; Fitness tradeoffs
200how did researchers learn myc is a small molecule, not a membrane bound protein?
They separated AMF spores from the plant with a membrane that only allowed small, mobiles signals to pass through and observed that the plant initiated colonization process without even touching the fungi.
201why use RNAi knockdown instead of gene knockout?
Sometimes genes are essential. If you knock them out, the organism can’t survive. By reducing, but not eliminating a gene’s function, you are able to manipulate an essential gene and observe a phenotype without killing your organism
202why use calmodulin?
natural sensor, modified it to produce visual signals with FRET. can detect rapid changes
203if Ca spikes were determined with Oregon green, why do extra analysis with FRET?
Enabled differentiation of the type of spikes in different cell types and how they differ (AFM vs RLS)