RNA interferance as new approaches for pest management Nabil Killiny Assistant professor Plant Pathology Citrus Research and Education Center
Outlines Introduction Mechanism Applications Conclusion
Introduction: The Central Dogma
In the early 1990s A number of scientists observed independently that RNA inhibited protein expression in plants and fungi. This phenomenon, identified but not understood. Posttranscriptional gene silencing PTGS or co-suppression in plant (Napoli et al., 1990; Campbell, 2005) Quelling in fungi (Romano and Macino in 1992) Neurospora crassa RNA interference (RNAi) in nematodes (Fire et al 1991) C. elegans Animal
Gene silencing in Cenorhabditis elegans dsrna can cause a systemic RNA-interference. RNAi lead to partial or complete loss-of-function of specific genes. Could be used to determine the biological roles of certain genes. If targets important gene could lead to mortality!!!
Definition RNAi (RNAi) is a mechanism that inhibits gene expression at the stage of translation of specific gene.
Role of RNAi in nature Gene regulations Genome maintenance Development Defense mechanism Viral infection Transposable elements. RNAi overview During RNAi Double-stranded RNAs cut into short doublestranded RNAs, s(small) i(interfering) RNA's, by an enzyme called Dicer. These then base pair to an mrna through a dsrnaenzyme complex. This will either lead to degradation of the mrna strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi
Gene regulations RNAi acts to regulate gene expression by two dicerdependent mechanisms sirna (small interfering) mirna (microrna)
The Players In Interference RNA sirna: dsrna 21-22 nt. mirna: ssrna 19-25nt. Encoded by non protein coding genome RISC: RNA induced Silencing Complex, that cleaves mrna Enzymes Dicer : produces 20-21 nt cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre mirna; which is later processed by Dicer
sirnas Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of posttranscriptional gene silencing mirna RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mrna and tag it for degradation A single base pair difference between the sirna template and the target mrna is enough to block the process. Originate from capped & polyadenylated full length precursors (pri-mirna) Hairpin precursor ~70 nt (pre-mirna) Mature mirna ~22 nt (mirna) Each strand of sirna has: a. 5 -phosphate termini b. 3 -hydroxyl termini c. 2/3-nucleotide 3 overhangs
Difference between mirna and sirna Function of both species is regulation of gene expression. Difference is in where they originate. sirna originates with dsrna. sirna is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. mirna originates with ssrna that forms a hairpin secondary structure. mirna regulates post-transcriptional gene expression and is often not 100% complementary to the target. And also mirna help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS)
Defense mechanism Schematic illustration of systemic viral spread as well as RNAi and subsequent viral recovery in plants. Green and red indicate the presence and loss of GFP fluorescence, respectively, and orange denotes the presence of both colors. The red dots on leaves show viral lesions. The bold arrows indicate the stages of plant growth, and the leaves are numbered accordingly. An arrow with a thin line shows a newly emerged leaf recovered from viral attack.
Understanding how RNAi works is the key to using it as a genetic tool and for pest control
Dicer is required for RNAi fluorescence dsrna wild-type dcr1 -/-
They used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles.
RNAi and the genes that control it in plants and animals. From RNAi for insect-proof plants Karl H J Gordon & Peter M Waterhouse Nature Biotechnology 25, 1231-1232 (2007) doi:10.1038/nbt1107-1231
Citrus Greening RNAi CLasinteractions Feeding activity Transmission factors Flight ability Healthy CLas-infected
Efficiency Application! Facing the challenges
Genes implicated in development (metamorphosis ) Topical application Gene/protein expression Morphology/biology Malformation Survival Flight ability Feeding ability X
Pauropsylline type nymph Psylline type nymph White and Hodkinson 1981 Triozine type nymph Diaphorina citri
I- IIItarget A Cloning B M PCR product containing the T7RNA Polymerase promoter sequences in the plus strand (A) and minus strand (B). M. DNA molecular markers. DNA template II- dsrna single strands Annealing the single stranded RNA plus and minus to create the dsrna
Wing malformation
Myosins comprise a family of ATPdependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes.
4 3.5 Gene expression 3 2.5 2 1.5 1 0.5 0 myl myh myl myh myl myh myl myh myl myh myl myh myl myh instar1 inster2 inster3 inster4 inster5 teneral adults Expression of myosin genes during ACP development
30 25 # of emerged adults 20 15 10 5 0 0ng 10ng 50ng 100ng 500ng 1000ng 0ng 10ng 50ng 100ng 500ng 1000ng 0ng 10ng 50ng 100ng 500ng 1000ng myl-dsrna myh-dsrna gfp-dsrna Effect of knocking down of myosin on ACP development
Survival probability Survival probability Survival probability Test Statistics Method Chi-Square DF P-Value Log-Rank 63.1915 5 0.000 Wilcoxon 40.2763 5 0.000 Test Statistics Method Chi-Square DF P-Value Log-Rank 63.0560 5 0.000 Wilcoxon 37.5827 5 0.000 Test Statistics Method Chi-Square DF P-Value Log-Rank 0.317882 5 0.997 Wilcoxon 0.398735 5 0.995 Lifespan (days) 16 14 12 10 8 6 4 2 0 Lifespan (days) 18 16 14 12 10 8 6 4 2 0 Lifespan (days) 18 16 14 12 10 8 6 4 2 0 dsrna-myl dsrna-myh dsrna-gfp Effect of knocking down of myosin on ACP fitness
1.2 1 0.8 0.6 0.4 Gene expression 0.2 0 1.2 1 0.8 0.6 tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh 0ng 10ng 50ng 100ng 500ng 1000ng myl-dsrna 1.2 1 0.8 0.6 0.4 0.2 0 0.4 0.2 0 tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh tubulin myl myh 1000ng gfp-dsrna 0ng 10ng 50ng 100ng 500ng 1000ng myh-dsrna
myh myl Hot plate (60 C) GFP water 0 5 10 15 20 # of jumps/ 10 sec
Control myl-dsrna myh-dsrna Ultrastructural microct images of D. citri adults showing the effects RNAi treatments. Two-dimensional coronal microct cross sections through the body and wing joints (white arrows) show that muscle tissue was not visible in healthy adults (A), but muscle groups were visible in adults treated with RNAi targeting light (D) or heavy chain (G) myosin. Three-dimensional volume renderings of ACP adults treated with RNAi targeting light and heavy chain myosin showed abnormal development of the wing attachment point compared to control adults. Close up images of the wing attachment (red highlighted area) revealed diaphanous and weakened connections to the wing (blue highlighted area) in adults treated with RNAi targeting light chain myosin, while connections were sunken and malformed in adults treated with RNAi targeting heavy chain myosin
Three-dimensional microct volume renderings of D. citri adults showing the internal ultrastructure of the thorax Muscles tissue are invisible in control Flight Muscles group are visible in dsrna treated insect
Controls LC-myosin HC-myosin Thorax Sagittal Traverse
Control Mechanism is complete MLC-dsRNA Brain keeps sending signals to make Ca+ available and bind to tropnin Troponin is saturated with Ca+ Relaxed muscles ( no Ca+) Muscles are not detected by X-ray Troponin is saturated with Ca+ Muscles are detected by X-ray
H 2 O gfp-dsrna Ca+ Bone Calcification In Foot calcium oxalate kidney stones myl-dsrna Relationship of Calcium Penetration and Contrast to Photon Energy
Control myl-dsrna Ca+ treated
Reduction in gene expression 1 0.1 0.01 dsrna concentration 0ng 50ng 100ng 500ng 1000ng awd myl myh troi Awd myl myh troi
RNAi and the genes that control it in plants and animals. From RNAi for insect-proof plants Karl H J Gordon & Peter M Waterhouse Nature Biotechnology 25, 1231-1232 (2007) doi:10.1038/nbt1107-1231
Schematic representation of the CTV genome in binary vector pcambia-1380 showing potential insertion positions (shown by upward arrows), where foreign genes could be engineered in the genome.
CTV p23 ORF tpds ttroponin tawd CTV 3 NTR CTV p23 ORF tpds/or ALAd twnt tmyocin CTV 3 NTR Various types of silencing inserts have been/will be tested with CTV-based silencing vector; they include (A) one gene fragment insertion with promoter (B) two gene fragments in-tandem insertion with promoter (C) one gene fragment insertion without promoter (D) two gene fragments in-tandem insertion without promoter (E) three gene fragments in-tandem insertion without promoter to enable silencing of more than one target at any one event.
Summary of agroinfiltration procedure of Citrus tristeza virus (CTV) vector constructs into Nicotiana benthamiana, CTV virion isolation and inoculation to citrus. Fully expanded true leaves of N. benthamiana plants were infiltrated with agrobacterium culture of CTV constructs in the binary vector plasmid. After 4-8 weeks post infiltration, systemic leaves of N. benthamiana leaves showing CTV symptoms are harvested. CTV virions are isolated by sucrose cushion gradient ultracentrifugation. Isolated virions are examined under electron microscope. CTV virions are inoculated to Citrus macrophylla plant by bark-flap method.
ACP wing development
A B C 1.0 0.8 Survival probability 0.6 0.4 0.2 0.0 0 5 Control dsrna 10 15 Days 20 25 D Average of adult lifetime 20 18 16 14 12 10 8 6 4 2 0 Control dsrna
Designing ideal Psyllid Trap Plants to protect and increase sustainability of new citrus plantings.
Carotenoid biosynthesis pathway
δ ALA dehydratase
δ ALA dehydratase
Abundance at 296 nm (mau) Chlorophyl B Lutein Chlorophyl A Zeaxanthin Control CTV-wt CTV-t-AWD CTV-t-δ ALA CTV-tPDS CTV-tPDS-As CLas-infected Cis β-carotene β-cryptoxanthin α-carotene β-carotene Isolutien Retention time (min)
Abundance at 296 nm (mau) Phytoene 1 Control CTV-wt CTV-t-AWD CTV-t-δ ALA CTV-tPDS CTV-tPDS-As CLas-infected Phytoene 2 Concentration (μg/g leaf tissue) Phyotene 1 ns Abundance Retention time (min) Phytoene 1 Phytoene 2 Concentration (μg/g leaf tissue) Phyotene 2 * Wavelength (nm) Wavelength (nm)
Carotin Carotin Phaeophytin A Phaeophytin B Chlorophyll A Lutein Chlorophyll B Violaxanthin Neoxanthin Lutein
Chlorophyl A Chlorophyl B Abundance Wavelength (nm) Concentration (μg/g leaf tissue a Chlorophyl A b a Chlorophyl B b
P< 0.0001 a b c d Number of D. citri settled per plant (%) P< 0.0001 P< 0.0001 P= 0.0003 P< 0.0001 P= 0.047 CTV-wt (control) CTV-tPDS-As CLas-infected
CLas infected CTV-tPDS-As CTV-wt (control) 0% 20% 40% 60% 80% 100% 120% Base Middele Top
Number of insects landed Number of insects landed Light Dark
Questions? What do you think?