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Leafhopper Comparative Genomics - IdentifyingSimilarities and Differences across LeafhopperVectors of Xylella fastidiosaAuthor(s) :E. W. Welch, W. B. Hunter, K. S. Shelby, R. F. Mizell,C. Tipping, C. S. Katsar and B. R. BextineSource: Southwestern Entomologist, 36(3):305-321. 2011.Published By: Society of Southwestern EntomologistsDOI:URL: http://www.bioone.org/doi/full/10.3958/059.036.0308

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VOL. 36, NO. 3 SOUTHWESTERN ENTOMOLOGIST SEP. 2011

Leafhopper Comparative Genomics - Identifying Similarities and Differences across Leafhopper Vectors of Xylella fastidiosa

E. W. Welch1, W. B. Hunter2, K. S. Shelby3, R. F. Mizell4, C. Tipping5, C. S. Katsar6,and B. R. Bextine1

Abstract. Insects in the order Hemiptera are considered the second most important group of plant pathogen vectors, after aphids as agriculture crop pests. Genomic approaches are providing new information on the genetic basis of biology, behavior, and refinement of their phylogenetic classification. Three leafhopper species, important as vectors of plant pathogenic bacteria referred to as Xylella fastidiosa (Hemiptera: Cicadellidae), were examined by comparison of the available expressed sequence tags, ~43,400 ESTs from three leafhopper species (Hunter datasets, NCBI). These species are vectors of the plant-pathogenic bacterium, Xylella fastidiosa (Wells et al.) the causal agent of Pierce’s disease of grapevine. A tentative look at the gene expression across these three leafhopper species, the glassy-winged sharpshooter, Homalodisca vitripennis (Germar), blue-green sharpshooter, Graphocephala atropunctata (Signoret), and black-winged sharpshooter, Oncometopia nigricans (Walker), were analyzed. After comparison approximately 4,800 specific transcripts for each species were obtained, with most of these (~40-48%) being identified as house-keeping. While the assembled datasets are not complete representations of all the leafhopper transcriptomes, these are predicted to be approximately one-fourth of the active genes in the genomes of these leafhoppers, based on comparative analysis of genomes of other insects in the order Hemiptera, based on an average of ~15k-25,000 active genes. Interest in host plant utilization led us to focus on a poorly studied set of transcripts from leafhoppers the desaturases. Delta-9 desaturase enzymes have been shown to be highly conserved throughout Eukarya (fungi, protists, plants, and animals) and specifically function to place double bonds between the adjacent carbons of specific fatty acids, playing a vital role in the internal metabolism and physiology of insects. The -9 desaturase sequences of several insect species, including the three leafhopper species of this study, were used to construct a phylogenetic tree. Additional analysis highlights differences for species-specific targeting of these genes within leafhoppers. It is proposed that as new developments in genomics and ________________________ 1Department of Biology, The University of Texas at Tyler, 3900 University Blvd., Tyler, TX 75799. 2USDA, ARS, U.S. Horticultural Res. Lab, 2001 South Rock Rd., Ft. Pierce, FL 34945. 3USDA, ARS, 1503 S. Providence, Res. Park, Colombia, MO 65203. 4University of Florida, IFAS, N. Florida, Res. and Education Center, 155 Res. Rd, Quincy, FL 32351. 5Delaware Valley College, 700 East Butler Ave., Doylestown, PA 18901. 6USDA, ARS, NPRU, 1011 Forrester Dr. SE., Dawson, GA 39842. Contact: Wayne.hunter@ars.usda.gov or BBextine@UTTyler.edu The use or mention of a trademark or proprietary product does not constitute an endorsement, guarantee, or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other suitable products.

305

strategies like RNA-interference emerge, researchers will be able to design specific and effective management tools to reduce leafhopper abundance, and/or transmission of disease by leafhoppers.

Introduction

Sharpshooter leafhoppers (Hemiptera: Cicadellidae) constitute a group of vectors of the plant pathogenic bacterium, Xylella fastidiosa (Wells et al.), which isthe causal agent of a number of economically important destructive plant diseases that reduce the production of grapes Vitis sp. (L.), peaches, Prunus persica (Batsch), Citrus varieties, and other fruit and woody ornamentals (Almeida et al. 2005a,b). Understanding how leafhopper physiology interacts with host plant utilization and how this may influence pathogen transmission is an important step toward the development of new management strategies to reduce crop losses associated with leafhopper transmitted diseases. Advances in genomics permits researchers to examine thousands of genes expressed during feeding, development, pathogen acquisition, and transmission (Hunter et al. 2003, Sabater-Munoz et al. 2006).

The examination of genes associated with feeding and digestion provides a better understanding of the digestive physiology and nutritional needs of leafhoppers (Coudron et al. 2007). Identification of proteins and peptides associated with leafhopper nutrition helps define those produced by leafhoppers versus those that are plant derived or associated with symbiotic bacteria which may aid leafhopper survival (Cohen 2002, Jain and Basha 2003, Rep et al. 2003).

The leafhoppers examined here, feed almost exclusively on the xylem fluid of plants (Andersen et al. 1989, 1992; Mizell et al. 2008). Xylem fluid is 98% water and nutrient-poor, containing several magnitudes fewer organic compounds than phloem or leaf tissue (Andersen et al. 1988). Despite the fact that organic constituents in xylem fluid often vary between plant species, the major organic constituents in xylem fluid are 19 amino acids, five to seven organic acids, and at least three sugars (Pate 1980; Andersen et al. 1989, 1992; Mizell et al. 2008). Because of the generally low concentration of nutrients in xylem tissue, xylem feeders such as leafhoppers must be efficient in the assimilation and utilization of the nutrients present. The glassy-winged sharpshooter, Homalodisca vitripennis(Germar), has shown 99% assimilation of amino and organic acids, along with the primary sugars found within xylem fluid (Mizell et al. 2008). Despite the fact that lipids are not found in high concentrations within plant xylem and therefore are not thought to be a large part of leafhopper diet, lipids have an essential role in leafhopper physiology and must be obtained through diet, or for the larger part, be generated through biosynthesis. All insect species rely on lipids for physiological processes. Lipids play an essential role in biological processes, namely production of tissues and oocyte development (Ziegler and Antwerpen 2006). Generally, the major fatty acid produced from fatty acid biosynthesis in insects, as well as in mammals and birds, is palmitic acid (16:0) which can be modified further by desaturases and other enzymes for various functions within the insect (Beenakkers et al. 1985).

Delta-9 desaturase-1 has been proposed to be a palmitoyl desaturase within glassy-winged sharpshooter (Hunter 2004), and similar -9 desaturases have been identified within black-winged sharpshooter, Oncometopia nigricans (Walker)

306

and blue-green sharpshooter, Graphocephala atropunctata (Signoret), with specificity of the latter two unknown. The sequence analysis of leafhopper desaturase may provide clues as to the degree of homology between these three species, the role of the enzymes within the internal metabolism of each species, and its effect, if any, on species adaptation to harsh environmental conditions as found in their overwintering range. Increased comprehension of leafhopper digestive physiology will identify the nutritional requirements, give clues to host range influences, and provides critical information needed for effective mass- rearing methods (Coudron et al. 2007).

Materials and Methods

Adult blue-green sharpshooters were obtained from a colony established by Dr. Alexander Purcell at the University of California, Berkeley. Founder blue-green sharpshooter were field-collected from mugwort (Artemisia douglasiana L.) in Guerneville, CA (Sonoma Co.) and subsequently reared on sweet basil (Ocimum basilicum L.) at 25°C (+10°C/-5°C), 14:10 L:D hours. First-generation progeny were macerated in RNAlater® RNA Stabilization Reagent (Ambion, Austin, TX) and stored at -40 C before shipment. Adult glassy-winged sharpshooters were collected from Citrus trees near Riverside, CA (Dr. Heather Costa). Adult black-winged sharpshooters were collected from crape myrtle (Lagerstroemia indica (L.) Pers.) of the loosestrife family, near Quincy, FL. Both of these species were collected and homogenized directly into RNAlater® RNA Stabilization Reagent (Ambion, Austin, TX). Total RNA extractions were as in Hunter et al. 2009.

Base calling was performed using TraceTuner™ (Paracel, Pasadena, CA), and low-quality bases (quality score <20) were stripped from both ends of each expressed sequence tag. Quality trimming, vector trimming, and sequence fragment alignments were executed using Sequencher™ software (Gene Codes, Ann Arbor, MI). Sequencher contig assembly parameters were set using a minimum overlap of 50 bp and 90% identity. Contigs joined by vector sequence were flagged for possible misassembly and manually edited. The -9 desaturase sequences obtained from each of the three species were aligned using Bioedit (Hall 1999), and conserved domains were identified. Further sequence identity was determined based on BLAST similarity searches using the NCBI BLAST server (www.ncbi.nlm.nih.gov) with comparisons made to both non-redundant nucleic acid and protein databases using BLASTN, BLASTX, and protein BLAST. Matches with an E-value -10 were considered significant and were classified according to the Gene Ontology (GO) classification system. Translated proteins were analyzed with National Center for Biotechnology Information’s BLASTp, Pfam (www.pfam.org), InterProScan (www.ebi.ac.uk), and Expert Protein Analysis System (www.expasy.org). A partial list of ~29 transcripts (Table 1) shows homologous matches between leafhoppers and the E-values showing relative identities.

The predicted leafhopper desaturase protein sequences identified were aligned using T-Coffee (www.tcoffee.org) and ClustalW (www.ebi.ac.uk/Tools/clustalw/), against a variety of homologs in different taxa. Alignments were retrieved and visualized in Treeview v1.6.6.

307

Results

The number of GWSS sequences available in Genbank (Hunter 2003, NCBI, EST), was analyzed against EST libraries produced to two other sharpshooter leafhopper species, Oncometopia nigricans (Hunter et al. 2004, 2005) and Graphocephala atropunctata (Hunter et al, 2006, 2007) NCBI EST’s, GenBank. The Total 44,300 EST sequences were analyzed using Blast2Go software analysis. Roughly 2,477 contigs were assembled and 3,681 singletons were produced for GWSS. The average length of the assembled contigs was 570 bp. Of the 9,860 EST from Oncometopia nigricans, a set of ~4,500 transcripts, with 1,807 contigs post assembly was obtained, with a set of ~4,830 transcripts, with 2,032 contigs post assembly of the 9,650 ESTs from Graphocephala atropunctata. NCBI BLASTX was used to find sequence similarities in GenBank for the assembled contigs and singletons. This resulted in a similar return of significantly identified sequences across all three leafhopper datasets using an E-value of >e-5 similar to findings of Hunter et al. 2009, EST analyses across genomes. As expected, the majority of these sequences corresponded to structural and housekeeping genes, but a great number correspond to genes of potential interest, such as desaturase, and others with potential as RNA interference targets, including genes for cuticle formation, development, hormones, eye morphogenesis, lipid and carbohydrate metabolism. Transcripts which are expressed in gut tissues and genes expressed specifically in salivary glands are of growing interest (Hunter et al. 2011). Experiments are underway to begin assessing these potential RNAi targets for management of leafhoppers and other hemipteran pests.

The category of Catalytic activity showed the greatest percentage of sequences having a related molecular function (Fig. 1). BLASTX with unassembled datasets as singletons produced the largest percentages of sequences belonging to the biological process category, from glassy-winged sharpshooter (Fig. 2). Blast2Go analysis with the requirement of at least 20 members, for Cellular Components, resulted in the largest percentage of sharpshooter expressed sequence tags within the ‘Lipid particle’ category (Fig. 3). Analysis of ESTs within the category of Molecular Function, when set to 50 members or more, resulted in the greatest number of sharpshooter sequences within the ‘Structural ribosomal’ category (Fig. 4). Phylogenetic comparison of the three leafhopper species -9desaturase protein sequences showed the blue-green sharpshooter as divergent from the other two species (Fig. 5). BLAST alignment of the three desaturase sequences shows 100% coverage and an E-value of 0.0 and between Homalodiscaand Oncometopia, and Graphocephala with 70% coverage, 9e-109 E-value when aligned across this region with glassy-winged sharpshooter. Homology between the three species using the overlapping sequences within the Molecular Function category, showed that sequence homology was greatest between Homalodisca and Oncometopia, with Graphocephala remaining as the more distant. This finding supports current taxonomy separating these leafhoppers (Fig. 1).

308

Tabl

e 1.

Par

tial

Com

paris

on

of

cDN

A’s

in

Thre

e Le

afho

pper

S

peci

es,

Hom

alod

isca

vi

tripe

nnis

, W

HH

C,

Gra

phoc

epha

la

atro

punc

tata

, WH

GA

, and

Onc

omet

opia

nig

rican

s, W

HO

N.

Anal

ysis

was

mad

e us

ing

Bla

stX,

val

ues

appr

oach

ing

zero

are

mor

e si

gnifi

cant

in s

eque

nce

iden

titie

s (re

d bo

x).

Gen

es w

ith m

ore

varia

bilit

y ar

e in

dica

ted

with

a b

lue

box.

S

eque

nce

hom

olog

y w

asgr

eate

r be

twee

n H

omal

odis

ca a

nd O

ncom

etop

ia t

han

betw

een

Gra

phoc

epha

la a

nd e

ither

Hom

alod

isca

or

Onc

omet

opia

. T

his

findi

ng s

uppo

rts c

urre

nt t

axon

omy

sepa

ratin

g th

ese

leaf

hopp

ers.

O

nly

a pa

rtial

lis

t is

sho

wn

for

sequ

ence

s w

ithin

Mol

ecul

ar

Func

tion

cate

gory

.

309

Fig.

1.

Com

posi

te fi

gure

sho

win

g di

strib

utio

n of

Hom

alod

isca

vitr

ipen

nis

trans

crip

ts a

cros

s ot

her s

peci

es (y

-axi

s le

ft), w

ith th

eto

p 6

spec

ies

hom

olog

ies

bein

g in

the

se in

sect

s w

hose

gen

omes

hav

e be

en c

ompl

eted

: D

roso

phila

mel

anog

aste

r, Ae

des

aegy

ptii,

Trib

oliu

m c

asta

neum

, Ano

phel

es g

ambi

ae, N

ason

ia v

itrip

enni

s, a

nd A

pis

mel

lifer

a. M

olec

ular

func

tions

of t

rans

crip

ts

have

the

gre

ates

t nu

mbe

r w

ith:

Cat

alyt

ic a

ctiv

ity =

1,9

45;

Bin

ding

= 1

,731

; an

d th

en t

rans

porte

r ac

tivity

= 5

05.

Bro

adca

tego

ries.

R

epre

sent

s ~2

3,00

0 ES

T’s

from

thr

ee c

DN

A lib

rarie

s, A

dults

, 5th

inst

ar,

and

Mid

gut,

H.

vitri

penn

is,

(Bla

st2G

Oan

alys

is).

310

Fig.

2.

Seq

uenc

e D

istri

butio

n:

Bio

logi

cal P

roce

ss.

BLA

STX

with

sin

glet

ons,

cat

egor

ies

had

to h

ave

at le

ast 7

0 m

embe

rs.

Rep

rese

nts

EST

’s fr

om th

ree

cDN

A lib

rarie

s, A

dults

, 5th

Inst

ar, a

nd M

idgu

t. H

omal

odis

ca v

itrip

enni

s, (

Bla

st2G

O a

naly

sis)

. H

ighe

st c

ateg

orie

s in

des

cend

ing

orde

r: R

espo

nse

to s

tress

227

; Pro

ton

trans

port

167;

Res

pons

e to

che

mic

al s

timul

i 157

; G

lyco

lysi

s 13

9; In

star

/pup

al d

evel

opm

ent 1

37; L

arva

l dev

elop

men

t 129

; Mes

oder

m d

evel

opm

ent 1

24; I

ntra

cellu

lar s

igna

ling

casc

ade

118;

Pro

teol

ysis

117

; O

ogen

esis

113

; B

ehav

ior

111;

Am

ino

acid

met

abol

ic p

roce

ss 1

11;

Pro

tein

s am

ino

acid

ph

osph

oryl

atio

n 10

9;

Neg

ativ

e re

gula

tion

of

cellu

lar

proc

ess

109;

C

ytok

ines

is

107;

D

NA

met

abol

ic

proc

ess

105;

M

onoc

arbo

xylic

aci

d m

etab

olis

m 1

04.

311

Fig.

3.

Seq

uenc

e D

istri

butio

n: C

ellu

lar C

ompo

nent

. C

ateg

orie

s ha

d to

hav

e at

leas

t 20

mem

bers

. R

epre

sent

s E

ST’s

from

th

ree

cDN

A lib

rarie

s, A

dults

, 5th

ins

tar,

and

Mid

gut.

Hom

alod

isca

vitr

ipen

nis,

(B

last

2GO

ana

lysi

s).

Gre

ates

t nu

mbe

r in

de

scen

ding

ord

er:

Lip

id P

artic

le =

509

; La

rge

ribos

omal

= 1

44;

Sm

all

Rib

osom

al =

125

; A

ctin

Fila

men

t =

97;

Tubu

lin

com

plex

= 7

3.

312

Fig.

4.

Seq

uenc

e D

istri

butio

n: M

olec

ular

Fun

ctio

n. C

ateg

orie

s ha

d to

hav

e at

leas

t 50

mem

bers

. R

epre

sent

s E

ST’s

from

th

ree

cDN

A lib

rarie

s, A

dults

, 5th

ins

tar,

and

Mid

gut.

Hom

alod

isca

vitr

ipen

nis,

(B

last

2GO

ana

lysi

s).

Gre

ates

t nu

mbe

r in

de

scen

ding

ord

er:

Stru

ctur

al r

ibos

omal

= 2

96; C

alci

um b

indi

ng =

218

; Mic

rofil

amen

t mot

or a

ctiv

ity =

180

; Pho

spho

ryla

tive

mec

hani

sm =

154

; End

opep

tidas

e ac

tivity

= 1

14; O

xido

redu

ctas

e ac

tivity

= 1

09; T

rans

crip

tion

regu

lato

r act

ivity

= 9

9.

313

Fig. 5. Tree based on 9 desaturase protein sequences of various insect species. Three leafhopper species are shown circled and accession numbers as follows: Homalodisca vitripennis (gi|46561748|gb|AAT01079.1|.) and Oncometopia nigrians(gi|53830704|gb|AAU95195.1|.) based on BLAST alignments performed through NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Another partial protein sequence identified as Graphocephala atropunctata showed 70% coverage and a 9e

-109 e-

value when aligned with H. vitripennis.

Discussion

While genomics and the power of bioinformatics analyses permit comparisons between species and across insect orders, there is still a need to increase the number of genomes completed for insect species, especially within the Hemiptera. Even so, with a small amount of genomic information a preliminary analysis can lead researchers to make rapid advances in studies of insect

314

phylogeny, physiology, and development. Of the categories analyzed one of largest percentages of leafhopper ESTs showed coding for lipid metabolism, or related processes, which was not surprising considering that lipids and lipid transport play vital roles in insect physiology. For example, the insect cuticle, can account for as much as 50% of the dry weight of an insect, and contains a number of layers containing lipid mixtures with lipid transport systems used for movement into these layers (Lockey 1985). Flying insects, including leafhoppers, use lipids as a source of energy with excellent storage capability, and the lipid content within insects has been shown to change over the developmental stages of insects requiring increased lipid synthesis, transport, and utilization. Significant amounts of lipids are also deposited into the oocyte during oogenesis to be used as energy for the embryo (Downer and Matthews 1976), with approximately 30-40% of the dry weight of an egg consisting of lipids (Ziegler and Antwerpen 2006).

Graphocephala, the blue-green sharpshooter, unlike the other two species is endemic to California (Almeida et al. 2005a), is notably smaller and prefers riparian habitats, unlike glassy-winged sharpshooter and black-winged sharpshooter, which are often found in cultivated crops, and both of which are native to a large portion of the southeastern United States (Mizell et al. 2008) and Florida (Adlerz 1980). Even with differences in body size and host plant range, these leafhoppers showed strong homologies to the number of sequences which could be identified, or which remained unclassified (unknowns or hypothetical) after in silico analyses. This parallels similar results for EST analyses in psyllids, another hemipteran, which demonstrated that psyllid EST’s in relation to their putative protein homologues (BLASTX) had the greatest overall similarity to the mosquito, A. aegypti (homology matches better than E-value e-10). However there was no significant difference when the EST dataset was compared across five genome databases: Caenorhabditis elegans, Drosophila melanogaster, Apis mellifera, Aedes aegypti,and Homo sapiens (Hunter et al. 2009) in the percentage distribution of sequences. Individual pairwise comparisons to the five genome databases resulted in similar distribution patterns of homology matches at each of four categories of E-values (ranges were from e-10 to e-20 to e-50 to e-100). This nearly identical separation of sequence data is most likely dependent in large part to the amount and type of known data within each respective genomic database. Having more genomes will undoubtedly provide better identification of true species specific sequences, but will also increase the numbers of identifiable sequences, which may be naturally similar across most organisms. As to the high rate of non-significantly matched sequences which estimates potential unique sequences within cDNA libraries this is most likely to be an overestimation due to several factors, such as computer alignment parameters, as well as low quality internal sequences. Moreover, assembled sequences may have lacked an open reading frame because they were too short causing cDNAs to consist mostly or entirely of a noncoding region (e.g., 3 untranslated region).

The -9 desaturase is found embedded in the membrane of endoplasmic reticulum and functions as either a palmitoyl or stearoyl -9 desaturase, placing a cis(Z) double bond at the ninth position of the carboxyl end of either 16:0 or 18:0 acyl CoA fatty acids, respectively (Watts and Browse 2000). The -9 desaturase 1 in glassy-winged sharpshooter was proposed to be a palmitoyl -9 desaturase, producing palmitoleic acid (16:1) (Hunter 2004). However, single -9 desaturases within Lepidoptera have been shown to catalyze the production of ratios of palmitoleic and oleic acids, raising an interesting point (Rosenfield et al. 2001), the

315

production of these acids in such ratios is thought to have a connection to pheromone production within Lepidoptera and other insect orders that use pheromones for mating; however, there has been no evidence to date that these leafhoppers produce pheromones. Comparisons within all of the -9 desaturases used in these analyses showed conservation of eight distinct histidine residues and three regions of conserved histidine cluster motifs that contain the residues HXXXXH, HXXHH, and EXXHXXHH, all essential for catalytic activity. Histidine residues like these, conserved at specific positions in all desaturases and many other di-iron proteins, act as the binding sites for iron, constituting the active site of the enzyme, and are highly conserved throughout Eukarya (Los and Murata 1998). All eight residues and the three regions mentioned were evident in leafhopper desaturase sequences. More genomic sequencing from leafhoppers will increase the identification of desaturases and other enzymes important for leafhopper biological functions. Unfortunately, leafhoppers as a group still have very little genomic information available.

The information gained from this study provides an early investigation using comparative genomics of the transcriptomes from three leafhopper vectors of Pierce’s disease of grapes: H. vitripennis, G. atropunctata, and O. nigricans. The increasing application of transcriptional data is leading the way in the development of new strategies to combat plant diseases and their insect vectors. Application of RNAi strategies against insects and other organisms are viewed as the future in insect pest control (Bellés 2010), and many new methods that incorporate delivery or expression of dsRNA within plants are being evaluated (Hunter et al. 2011). Collectively, these genetic sequences provide the foundation needed for further functional genomics studies that will enable the development of more biorational management strategies to reduce losses to disease pathogens spread by these and other leafhopper pests.

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