M o l e c u l a r P l a n t Pathology (2 0 2 0 ) 2 1(1 ), 1 0 9 – 1 2 3
DOI: 10.1111/mp p. 12884
The flagella of ‘Candidatus Liberibacter asiaticus’ and its movement
in planta
M AXUEL O. A NDRADE 1 , Z HIQIAN PANG 1 , D IA NN S. AC H O R 1 , H A N WA NG 1 , T INGS H A N YAO 1,2 ,
1,
*
B URTON H. SINGER 3 AND NIAN WANG
1
citrus research and education center, Department of Microbiology and cell Science, university of Florida/Institute of Food and agricultural
Sciences, lake alfred, Fl, uSa
2
national engineering research center for citrus, citrus research Institute, Southwest university, chongqing, 400712, People’s republic of china
3
emerging Pathogens Institute, university of Florida, gainesville, Fl, uSa
SUMMARY
INTRODUC TION
Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is
the most prevalent HLB causal agent that is yet to be cultured.
Here, we analysed the flagellar genes of Las and Rhizobiaceae
and observed two characteristics unique to the flagellar proteins
of Las: (i) a shorter primary structure of the rod capping protein
FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only
one flagellin-encoding gene flaA (CLIBASIA_02090), whereas
other Rhizobiaceae species carry at least three flagellin-encoding
genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays
demonstrated that FlgJLas interacts with FlgB but not with FliE.
Ectopic expression of flaALas in A. tumefaciens mutants restored
the swimming motility of ∆flaA mutant and ∆flaAD mutant, but
not that of the null mutant ∆flaABCD. No flagellum was observed
for Las in citrus and dodder. The expression of flagellar genes
was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses
flagellin protein in psyllids, but not in planta. The flagellar features
of Las in planta suggest that Las movement in the phloem is not
mediated by flagella. We also characterized the movement of Las
after psyllid transmission into young flush. Our data support a
model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las
out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.
Citrus huanglongbing (HLB, also called citrus greening) is the most
devastating citrus disease worldwide. The most prevailing HLB
pathogen in the world is ‘Candidatus Liberibacter asiaticus’ (Las),
an unculturable bacterium vectored by Asian citrus psyllid (ACP,
Diaphorina citri), which is an invasive pest for citrus-producing areas
outside of Asia (Bové, 2006; Wang and Trivedi, 2013). Currently, HLB
management strategies mainly rely on psyllid control and numerous horticultural approaches (Blaustein et al., 2018; Li et al., 2016,
2017, 2018) since all commercial citrus varieties are susceptible to
HLB (Folimonova et al., 2009). Current HLB and ACP management
has not prevented HLB from spreading worldwide (Wang, 2019).
For example, HLB has spread throughout Florida, to other major
citrus-producing states (e.g. California and Texas) in the USA, and
to neighbouring countries including Mexico and countries in the
Caribbean and Central America (Wang et al., 2017). It is paramount
to understand the biology and virulence mechanism of Las to design
a suitable and efficient HLB control strategy. However, its unculturability has hampered our investigation of the biology and virulence
mechanism of Las. Its close relatives, such as Liberibacter crescens,
Agrobacterium and Sinorhizobium, have been used as surrogates
in previous studies (Andrade and Wang, 2019; Jain et al., 2019;
Naranjo et al., 2019; Vahling-Armstrong et al., 2012).
Las has been observed to be distributed throughout the
whole plant, including leaf, stem, root, flower and seed coat
(Tatineni et al., 2008). However, the details of Las movement in
planta are still unknown. Most bacteria possess more than one
organelle for motility. For most bacteria, flagella are the major
motility organelles responsible for swimming and swarming,
whereas type IV pili are responsible for the twitching motility. Las encodes the type IVc tight adherence-pili (Tad) that are
usually not associated directly with motility. Instead, they are
involved in adherence (Andrade and Wang, 2019). Flagellummediated motility allows bacterial cells to move toward and explore nutrient-rich habitats and move away from unfavourable
environments (Anderson et al., 2010; Berry and Armitage, 1999;
Keywords: citrus, flagella, HLB control, huanglongbing,
Liberibacter, movement, psyllid.
*Correspondence: Email: nianwang@ufl.edu
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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
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provided the original work is properly cited.
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M.O. ANDRADE et al.
Moens and Vanderleyden, 1996; Zhu et al., 2013). Flagella
also play a key role in surface attachment and host–bacteria
interactions (Heindl et al., 2014; Rossez et al., 2015). It has
been known that some flagellated plant pathogenic bacteria
suppress expression of flagella in the plant, probably to avoid
triggering plant defence (Chatnaparat et al., 2016; Yu et al.,
2013). Las contains 30 flagellar genes located in three clusters on the chromosome (Fig. 1) (Duan et al., 2009). However,
it remains to be determined whether Las synthesizes flagella
during within-plant movement. This is a fundamental question
that has clear implications for the rate at which disease symptoms appear in both new flush and old leaves.
The flagellar machinery can be divided into five parts called
the basal body, the hook, the hook–filament junction zone, the filament and the filament cap (DeRosier, 2006; Evans et al., 2014;
Ghosh, 2004; Minamino and Namba, 2004). The basal body is
composed of the cytoplasmatic C-ring (FliG, FliM and FliN), the
inner or MS-ring (FliF), the periplasmatic P-ring (FlgI), the outer
membrane L-ring (FlgH), the proximal rod (FlgB, FlgC, FlgF and
FliE) and the distal rod (FlgG) (Jones and Aizawa, 1991; Park
et al., 2006). The basal body is embedded in the cell surface and
plays a role in flagella rotation together with the Mot proteins
(Minamino et al., 2008). The hook and filament, which are tubular
structures composed of subunits of hook (FlgE) and flagellin (FliC),
respectively, extend outwards from the cell (Macnab, 2003). The
hook length and the switch of specificity from substrates hook-tofilament are tightly regulated by FliK (Erhardt et al., 2010; Waters
et al., 2007). Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks), lack of filament structures and nonmotile
phenotypes (Minamino et al., 2009). The hook–filament junction
zone (FlgK and FlgL) and the filament cap (FliD) are located between the hook and filament and at the tip of the filament, respectively (Ikeda et al., 1996). Flagellar assembly begins with the
basal body, proceeds with the hook and finishes with the filament.
Capping proteins (FlgJ, FlgD and FliD) are needed to permit and
regulate the polymerization of rod, hook and filament, respectively,
at different stages of flagellar assembly (Minamino and Namba,
2004). FlgJ, FlgD and FliD exist at the tip of the growing rod, hook
and filament structures, respectively (Evans et al., 2014).
The flagellar export apparatus is built into the central pore
of the basal body MS-ring, and it is formed by interaction of
six membrane proteins: FlhA, FlhB, FliP, FliO, FliQ and FliR, and
three cytoplasmic proteins FliH, FliI and FliJ. FliI is the ATPase that
provides the energy for the translocation of proteins across the
cytoplasmic membrane (Macnab, 2003). FliH acts as an ATPase
regulator, coupling the energy of ATP hydrolysis to flagellar protein export (Evans et al., 2014; Macnab, 2003). Also, the chaperone, FliJ, prevents premature aggregation of substrates in the
cytoplasm, and FlgA, assists the P-ring assembly in the periplasm
(Evans et al., 2006; Macnab, 2003).
Flagellins are the subunits that compose the flagellar filament
and represent the major flagellar structural protein (Altegoer
et al., 2014; Rossez et al., 2015). The number of flagellin-encoding
genes in bacterial genomes varies and may range between one
(Escherichia coli) and seven (Vibrio parahaemolyticus) (Fedorov
and Kostyukova, 1984; Kim and McCarter, 2000; Stewart and
McCarter, 2003). The flagellar filaments in E. coli K-12 are built
up from only one flagellin protein (FliC) (Turner et al., 2012),
whereas Bdellovibrio bacteriovorus (Thomashow and Rittenberg,
1985), Campylobacter coli (Guerry et al., 1990), Caulobacter crescentus (Driks et al., 1989), Agrobacterium tumefaciens (Deakin
et al., 1999; Mohari et al., 2018), Rhizobium lupini strain H13-3
and Sinorhizobium meliloti have at least two flagellin proteins
forming their flagellar filaments (Scharf et al., 2001).
Young flush has been shown to be critical for ACP and Las
infection. ACP preferentially feeds and exclusively reproduces on
young, newly emerged flush shoots of citrus. ACP nymphs feed and
complete their life stages on young flush shoots. This observation
Fig. 1 Genetic organization of the three clusters encoding flagellar genes in the ‘Candidatus Liberibacter asiaticus’ genome. The names of the coding regions shown
in the diagram are based on their homology with flagellar genes in Rhizobiaceae bacteria. Grey arrows, conserved hypothetical protein; white arrows, hypothetical
proteins.
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Las flagella and movement
appears to be universal across different citrus-producing regions
worldwide (Bové, 2006; Hall et al., 2013; Setamou et al., 2016;
Sétamou and Bartels, 2015; Tomaseto et al., 2016). Not surprisingly, citrus trees are normally infected when new flush is present
(Hall et al., 2016). In addition, ACPs that acquire Las as adults are
poor vectors of Las compared with ACPs that acquire the pathogen as nymphs (Pelz-Stelinski et al., 2010). A threshold infection
level (c. 106 Las/psyllid) is required for successful transmission to
citrus plants (Ukuda-Hosokawa et al., 2015). How Las moves after
psyllid transmission remains largely unexplored.
Here, we investigated the flagella of Las. Our results indicate
that Las flagellar genes express at low levels in planta. Las moves
to other sink tissues with phloem sap when young leaves mature
into sources. This delayed Las movement after ACP transmission
into young flush might present opportunities to develop new
strategies for HLB management.
R E S U LT S
Comparison of flagellar genes of ‘Ca. Liberibacter’ and
other members of Rhizobiaceae
The analysis of the flagellar components encoded by the Las genome (Fig. 1 and Table 1) showed the following characteristics
that are shared by all bacterial species in Rhizobiaceae: (i) lack
of cytoplasmic chaperone FliJ and the components of export apparatus FliH/FliO; (ii) the function of FliK is performed by MotD
(Eggenhofer et al., 2006); (iii) the rod-capping FlgJ protein lacks
the muramidase domain in its carboxy-terminus (Herlihey et al.,
2014); and (iv) lack of FliD, the filament cap (Table 1). It is important to note that all the traits cited above are shared by members
of Rhizobiaceae that are known to contain flagellar apparatus
and show swimming behaviour, such as A. tumefaciens and
S. meliloti (Attmannspacher et al., 2005; Deakin et al., 1999;
Mohari et al., 2018).
We observed two characteristics unique to the flagellar proteins of Las and Liberibacter crescens BT-1, a culturable bacterium, that may determine the fate of the Las flagellar machinery:
1. It has a shorter primary structure of the rod-capping protein
FlgJ than other Rhizobiaceae bacteria (Fig. 2). In previous
work, it was shown that the minimal functional region
required of FlgJ for promoting the rod assembly consists
of 151 residues in its amino terminus (Hirano et al., 2001).
The primary structure of FlgJ in Las (CLIBASIA_01980) contains 112 amino acid residues, whereas in A. tumefaciens
(Atu0584) and S. meliloti (SMc03071), FlgJ homologues
consist of 175 and 184 residues, respectively.
2. Las contains only one flagellin-encoding gene flaA
(CLIBASIA_02090), whereas other Rhizobiaceae species
carry at least three flagellin-encoding genes (Fig. 1, Table 1).
For example, Rhizobium lupini H13-3 and S. meliloti produce
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flagellar filaments composed of three and four flagellin
subunits, respectively (Scharf et al., 2001). These flagellinencoding genes are named flaA through flaD. Indeed, the complex flagellar filaments in Rhizobiaceae bacteria are composed
of a majority flagellin and at least one secondary flagellin.
Mutational analysis of flagellin genes revealed that, in both
R. lupini and S. meliloti, FlaA is the principal flagellin and that
FlaB, FlaC and FlaD are secondary (Deakin et al., 1999). In this
way, FlaA and at least one secondary flagellin are required for
assembling a functional flagellar filament in R. lupini and S. meliloti (Scharf et al., 2001). Similar results were demonstrated in
A. tumefaciens (Mohari et al., 2018). Thus, the unique features
of Las in FlgJ and FlaA are probably critical to functionality of
its flagella.
Characterization of the function of FlgJ and FlaA of Las
using Agrobacterium as a surrogate
We first evaluated whether FlgJ and FlaA of Las are functional
by assessing whether they can complement corresponding mutants of A. tumefaciens (Atu). For this purpose, we constructed a
nonpolar mutant of flgJ in A. tumefaciens. As expected, the flgJ
mutant of A. tumefaciens became immobile, which is similar to
the ∆flaABCD mutant with all four flagellin genes deleted (Fig. 3).
Only flgJAtu but not flgJLas restored the swimming motility of
A. tumefaciens flgJ mutant (Fig. 3A,B), indicating flgJLas might
not be functional or works differently in Las. To verify whether
FlgJLas can physically interact with the proximal rod proteins FlgB
and FliE as shown in other well-studied systems (Hirano et al.,
2001), we performed a pull-down experiment with those proteins.
Interestingly, FlgJLas interacted with FlgB but not with FliE (Fig. 4).
To test the function of flagellin protein FlaALas, we generated
mutant strains of A. tumefaciens carrying deletion in flaA alone
(∆flaA), in flaA and flaD genes (∆flaAD), and in all flagellinencoding genes (∆flaABCD). Interestingly, ectopic expression of
flaALas in A. tumefaciens mutants restored the swimming motility
of ∆flaA mutant (Fig. 3C) and ∆flaAD mutant (Fig. 3D), but not
that of the null mutant ∆flaABCD (Fig. 3D). The swimming halos
for the tested strains were calculated and the means and standard deviation (n = 3) values are shown in Fig. S2. These results
indicate that in the A. tumefaciens flagellar system FlaALas functions as the majority flagellin interacting with other secondary
flagellins such as FlaB or FlaC, which remain intact in the ∆flaAD
mutant, to form a fully functional flagellar filament. However,
FlaALas alone could not form a fully functional flagellar filament
in A. tumefaciens.
Observation of Las flagellum in psyllids and in planta
To verify whether Las forms a functional flagellum, we visualized Las cells isolated from Las-infected plants and psyllids using
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Table 1
Identity of residues of flagellar proteins encoded by Rhizobiaceae bacteria (as percentage values)
Gene
Las
Atu
Sme
Lcc
Laf
Lso
+
46
45
54
81
82
Basal body
FliG
FliM
+
30
30
42
70
66
FliN
+
47
50
50
77
74
FliF
+
49
50
56
78
75
FlgI
+
64
64
68
85
87
FlgH
+
54
56
63
80
76
FlgB
+
45
47
57
76
75
FlgC
+
59
62
68
79
85
FlgF
+
44
46
49
77
75
FliE
+
37
37
42
67
63
FlgG
+
59
62
69
88
90
FlgJ
+*
29
32
37*
61*
68*
MotA
+
62
58
64
60
63
MotB
+
31
32
36
65
75
MotC
+
29
27
34
65
60
+
50
41
58
75
74
Hook
FlgE
FliK/MotE
+
32
28
29
44
48
FlgD
+
43
44
57
85
75
FlgK
+
36
36
47
76
69
FlgL
+
27
30
39
61
57
Junction zone
Filament
FlaA
+
50
42
49
67
61
FlaB
†
+
+
−
−
−
FlaC
†
+
+
−
−
−
FlaD
†
+
+
−
−
−
†
†
†
†
†
†
+
66
65
68
86
82
Capping filament
FliD
Export apparatus
FlhA
FlhB
+
44
45
54
80
74
FliP
+
60
57
67
90
86
FliO
†
†
†
†
†
†
FliQ
+
57
61
65
90
90
FliR
+
40
43
51
81
78
FliH
†
†
†
†
†
†
FliI
+
62
63
68
87
84
(Continues)
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Las flagella and movement
Table 1
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(Continued)
Gene
Las
Atu
Sme
Lcc
Laf
Lso
FliJ
†
†
†
†
†
†
FlgA
+
42
46
53
70
69
Chaperones
Note: Comparative analysis of flagellar proteins encoded by different species of Rhizobiaceae bacteria. Las, ‘Candidatus Liberibacter asiaticus’ (CP001677); Laf,
‘Candidatus Liberibacter africanus’ (CP004021); Lso, ‘Candidatus Liberibacter solanacearum’ (CP002371); Lcc, Liberibacter crescens strain BT-1 (CP003789); Atu,
Agrobacterium tumefaciens (AE007869); Sme, Sinorhizobium meliloti strain 1041 (AL591688).
*Indicates a shorter primary structure for FlgJ homologues when compared to A. tumefaciens and S. meliloti. †No homologues were identified. + and − indicate presence and absence of a homologue in the analysed genome, respectively.
Fig. 2 Sequence alignment of the rod cap proteins (FlgJ) encoded by Rhizobiaceae bacteria showing the rod-binding domain highlighted in green. Atu,
Agrobacterium tumefaciens (AE007869); Sme, Sinorhizobium meliloti strain 1041 (AL591688); Rhf, Sinorhizobium fredii NGR234 (CP001389); CLas, ‘Candidatus
Liberibacter asiaticus’ (CP001677); CLaf, ‘Candidatus Liberibacter africanus’ (CP004021); CLso, ‘Candidatus Liberibacter solanacearum’ (CP002371); Lcc, Liberibacter
crescens strain BT-1 (CP003789); CLam, ‘Candidatus Liberibacter americanus’ (CP006604). Alignment result was obtained by ClustalW and protein domain was
identified with Pfam.
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M.O. ANDRADE et al.
Fig. 3 Functional analysis of FlgJ and FlaA of ‘Candidatus Liberibacter asiaticus’ (Las) via complementation analysis of their corresponding mutants of
Agrobacterium tumefaciens (Atu) with swimming assay. (A) Complementation of A. tumefaciens flgJ mutant with flgJAtu.. (B) Complementation of A. tumefaciens
flgJ mutant with flgJLas. (C) Complementation of A. tumefaciens flaA mutant with flaALas and flaAAtu. (D) Complementation of A. tumefaciens flaAD and flaABCD
mutants with flaALas . WT, A. tumefaciens strain C58; ∆flaABCD, flagellum null mutant; ∆flgJ, A. tumefaciens C58 with deletion of flgJ; ∆flaA, A. tumefaciens C58
with in-frame deletion in flaA; ∆flgJ (flgJAtu), flgJ mutant complemented with A. tumefaciens flgJ; ∆flgJ (flgJLas ), flgJ mutant complemented with Las flgJ; ∆flaA
(flaAAtu), flaA mutant complemented with A. tumefaciens flaA; ∆flaA (flaALas), flaA mutant complemented with Las flaA; ∆flaA (EP), ∆flaA carrying empty plasmid;
∆flaAD, A. tumefaciens C58 with in-frame deletion for the flagellin-encoding genes flaA and flaD; ∆flaABCD, A. tumefaciens C58 carrying deletion for all flagellin
genes flaA, flaB, flaC and flaD; ∆flaAD (flaALas) and ∆flaABCD (flaALas), mutants ∆flaAD and ∆flaABCD harbouring the flagellin-encoding gene flaA from Las.
Bacterial cells were incubated at 28 °C and photographed after 3 days.
transmission electron microscopy (TEM). For the visualization,
Las cells were confirmed using Las-specific antibody against Las
OmpA and the second antibody anti-rabbit IgG-gold. Las cells
isolated from grapefruit (Citrus paradisi) seed coats (Fig. 5A)
and parasitic dodder stems (Cuscuta sp.) and psyllids are all
filament-shaped or long-rod-shaped. Previous observation of Las
under TEM suggested that it is polymorphic with Las cells being
mostly filamentous in psyllids, but in different shapes in plants
with diameter 0.33–1.5 μm and length 2.6–6.3 μm (Hartung
et al., 2010). No flagellum was observed for Las cells isolated
from grapefruit seed coats (Fig. 5A) and parasitic dodder stems
(Fig. 5B). For Las isolated from psyllid guts, no flagellum was observed on most Las cells. A few Las cells showed thread-like structures from which we could not make a conclusive claim whether
they are indeed flagella (Fig. 5C-H). However, L. crescens, which
shares the same flagellar genes as Las (Table 1), was observed to
contain flagella under TEM (Fig. 6).
Expression of flagellar genes in psyllids and in planta
To further assess whether Las synthesizes flagella in psyllids and
plants, we tested the expression of flagellar genes at both transcription and translation levels using quantitative reverse transcription
PCR (RT-qPCR). We found that flagellar genes were up-regulated in
psyllids compared to in planta (Fig. 7). Specifically, the flagellin gene
flaA is highly up-regulated in psyllids, which is consistent with our
previous study (Yan et al., 2013). Likewise, flagellin protein was detected for Las inside psyllids, but not inside plants based on western
blot (Fig. 8). Taken together, our data suggest Las lacks flagella in
planta or flagellar genes express at low level in planta.
Movement of Las in planta after ACP transmission of Las into
young flush
Las has been known to translocate through the sieve pore to move
from the infection sites to tissues throughout the plants (Kim et al.,
2009). Since Las does not produce flagella or its flagellar gene expression is low in planta, its movement to other parts (e.g. roots) of
the plant from the infection site is unlikely to be mediated by flagella.
Instead, we hypothesize that Las moves with phloem sap to sink
tissues when young flush matures. To test this hypothesis, we caged
ten Las-positive ACPs/flush (Valencia sweet orange) for each plant
for different durations and tested for Las at 54 days post-psyllid
feeding. For each treatment, we used five plants with one plant as
a biological replicate. After 54 days, 40%, 100%, 100% and 80%
of tested leaves were Las-positive following 6, 11, 18 and 29 days
of ACP feeding, respectively. However, none of the root samples
were Las-positive, indicating that Las had not moved to roots.
To further understand Las movement inside plants after ACP
transmission, we trimmed nine citrus plants (Hamlin sweet orange
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Las flagella and movement
Fig. 4 Pull-down assay showed a physical interaction between FlgJLas and
FlgB. (A) The protein GST-FlgJLas was used as a bait for the pull-down assay.
GST-FlgJLas was expressed in Escherichia coli, immobilized and washed on
glutathione sepharose beads, and incubated with E. coli lysates containing
6×HisEGFP, 6×HisFlgB or 6×HisFliE. Pull-down inputs containing the
supernant of total cell extracts (A) and (B) were immunobloted using the antiGST and anti-6×His antibodies, respectively. Eluted protein fractions (Elutes)
were probed with anti-6×His antibodies.
on Cleo) to trigger young flush. We caged 15 psyllids/plant together
with young flush and five mature leaves. Las was detected in
young flush-developed leaves in five of the nine plants at 4 weeks
after ACP transmission, and 7, 9 and 15 weeks after ACP transmission. Las was detected in the roots in five of the infected plants at
15 weeks after ACP transmission, but not at other time points. Las
was not detected in all the old leaf samples inside or outside of the
cage at the aforementioned time points. Taken together, our data
are consistent with our hypothesis that Las moves from source to
sink passively with phloem sap and will likely stay in the young
flush after ACP transmission and before flush matures.
DISCUSSION
Las flagella have multiple important features. First, FlgJ of
Las is not able to complement the flgJ mutant of A. tumefaciens. FlgJ plays an important role in flagellar assembly (Hirano
et al., 2001). FlgJLas, as its homologues in several other bacterial phyla, including Alphaproteobacteria, Deltaproteobacteria,
Epsilonproteobacteria and Spirochaetes (Nambu et al., 2006),
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contains only the rod-capping domain involved in protein–protein
interactions, but lacks the peptidoglycan hydrolase domain, which
allows the elongating flagellar rod to penetrate through the peptidoglycan (PG) layer. The peptidoglycan hydrolase domain is commonly found in FlgJ from other bacteria but its function can be
developed by other peptidoglycan hydrolase protein not related
to flagellar proteins. Liberibacter genomes code for one peptidoglycan hydrolase, which contains a soluble lytic transglycosylase (SLT) domain and a periplasmic signal peptide (Fig. S1). In
enteric bacteria such as Salmonella enterica serovar Typhimurium,
flgJ null mutants fail to produce the flagellar rods, hooks and
filaments, and are nonmotile (Hirano et al., 2001; Nambu et al.,
1999). Instead, the flgJ mutant of Borrelia burgdorferi, which contains a single-domain FlgJ homolgue as Las, can form intact flagellar basal bodies but had fewer and disoriented flagellar hooks
and filaments (Zhang et al., 2012). Accordingly, the motility of
the flgJ mutant of B. burgdorferi was partially deficient. In addition, FlgJ seems to be functional because it was able to interact
with FlgB. Thus, Liberibacter spp. may have evolved some novel
means involving FlgJ to assemble the rod. Second, even though
Las encodes a functional FlaA that complements the corresponding mutant of A. tumefaciens, the complex flagellar filament in
Rhizobiaceae bacteria is formed by the interaction of FlaA with at
least one secondary flagellin, such as FlaB, C or D, that is absent in
Las. Ectopic expression of FlaALas in ΔflaABCD did not restore the
motility of the null mutant, suggesting that FlaALas is incapable of
forming a fully functional filament. Alternatively, the observation
of flagella in L. crescens suggests synthesis of Las flagella related
to FlgJ and FlaA might involve some novel mechanisms different
from known examples in Rhizobiaceae. Lastly, even though we
could not totally rule out the possibility that Las might contain flagella in certain conditions (such as in psyllids, as implied in Fig. 8),
expression of the flagellar genes is repressed in planta. Indeed,
western blotting was not able to detect flagellin in planta.
The flagellar features of Las in planta might help Las
avoid the elicitation of plant defence responses since flagellin is known to trigger plant immunities as typical pathogenassociated molecular patterns (Thomma et al., 2011). Interestingly,
mammalian cells use different receptors for recognition of extracellular flagellin or intracellular flagellin. For sensing flagellin outside the mammalian cell, the immune system uses Toll-like receptor
TLR-5 (Yoon et al., 2012). If flagellin protein is detected within the
cytosol of a cell, it is detected by the Nod-like receptor (NLR) Ipaf
(Franchi et al., 2006). We reason that flagellin might activate some
early immune responses when Las is transmitted into the phloem
by psyllids, during which the flagellin expressed in psyllids remain
available in planta. It is likely the recognition is not mediated by
FLS2 (Gómez-Gómez and Boller, 2002), but instead by some unknown intracellular receptors. However, it is expected flagellar
expression is turned off soon after transmission. Interestingly, multiple plant pathogenic bacteria, such as Pseudomonas syringae
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Fig. 5 Transmission electron microscopy analysis of ‘Candidatus Liberibacter asiaticus’ (Las). (A) Las cell isolated from Las-infected grapefruit seed coat. (B) Las cell
isolated from dodder stem. (C)–(H) Las isolated from psyllid guts. (D), (F) and (H) are enlarged views of the indicated area in (C), (E) and (G), respectively. Las cells
were visualized by using negative staining and antibody against Las OmpA and second antibody anti-rabbit IgG-gold. Arrows indicate thread-like structures. Size bars
are indicated in the images.
Fig. 6 Visualization of the flagellar structures in Liberibacter crescens
BT-1 cells. Negatively stained cells were analysed by transmission electron
microscopy. Size bars are indicated in the images.
(Cheng et al., 2018; Markel et al., 2016) and Xanthomonas
axonopodis pv. glycines (Chatnaparat et al., 2016), have been
known to use a similar strategy by reducing expression of flagellar
genes and losing flagella once reaching their habitats inside the
plant. On the other hand, the higher expression of flagellar genes
in psyllids suggests flagella might be important for its movement
inside the psyllid body to different organs and cell organelles
(Ghanim et al., 2017). The induction of flagellar genes in psyllids
probably results from differential physiochemical conditions inside
psyllids from the phloem sap. In addition, the ACP genome encodes
a deficient innate immune system lacking a number of genes that
encode for antimicrobial peptides and the Imd pathway associated
with defence against Gram-negative bacteria (Arp et al., 2017).
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Fig. 7 RT-qPCR analysis of selected ‘Candidatus Liberibacter asiaticus’
(Las) genes encoding flagellum apparatus. Fold change is the relative gene
expression (in psyllid versus in planta) of Las.
We have proposed the following movement model for Las
after psyllid transmission into young flush (Fig. 9). In Phase 1 (lag
phase), Las initially undergoes no or slow growth to adapt to the
environmental changes from psyllids to phloem, then begins multiplication inside the young flush after an undefined period (Phase
2/multiplication phase). In the first two phases, Las stays inside the
young flush before it turns into a source. With the maturation of
the young flush, the infected leaf transits from a sink into a source.
Meanwhile, Las begins to move from the infected leaf to other
parts of the plant, such as roots, following phloem sap from source
to sink (Phase 3/movement phase). The Las flagellar features probably suggest that Las moves with phloem sap rather than relying
on flagella-mediated movement. This model is consistent with the
data that newly infected young leaves become infectious within
10–15 days after an inoculum of Las from an adult psyllid (Lee
et al., 2015). This movement pattern also promotes acquisition
and transmission of Las by nymphs. Las has been suggested to
move at approximately 2–3 cm/day in citrus after graft inoculation
(S. Lopes, personal communication). However, we do not think that
detecting Las in the leaves but not in the root results from the slow
movement of Las. The average height (top to root) of the trimmed
trees used for our Las movement assay is approximately 20 cm and
we conducted the qPCR analysis at 54 days after psyllid feeding,
which is supposed to be enough for Las to reach the root if moving right after feeding. In addition, microscopic analysis of Las in
planta showed that Las cells float freely in the phloem sap without
attaching to the sieve tube cell walls or each other (i.e. without
forming biofilms) (Ding et al., 2018; Folimonova and Achor, 2010;
Hartung et al., 2010; Hilf et al., 2013; Kim et al., 2009). A similar phenomenon has been observed for Lso and ‘Ca. Liberibacter
americanus’ in planta (Liefting et al., 2009; Secor et al., 2009; Wulff
et al., 2014). The bacteria move primarily in the vertical direction
along the sieve tubes through the sieve pores, rather than horizontally to adjacent sieve tubes. This hypothesis was first suggested by
observations of the uneven colonization of Las in dodder, where
Fig. 8 Flagellin detection. The antibody produced using the peptide
of ‘Candidatus Liberibacter asiaticus’ (Las) flagellin protein (sequence:
DRVSSGLRVSDAAD) was used to detect flagellin protein in different bacterial
samples (left to right): Agrobacterium tumefaciens C58 derivative strain;
Las-infected citrus (Ct = 28.23 ± 0.23); Las-free citrus control; Las-infected
psyllids (Ct = 26.89 ± 0.49); Las-free psyllid control; positive MBP-Las flagellin
fusion protein control and negative MBP protein control. The band marked
with an asterisk is the flagellin protein of Las with MW 48 kDa. Coomassie
brilliant blue (CBB) served as a loading control. The experiment was repeated
twice with similar results.
adjacent phloem vessel elements were observed to be completely
full of Las or free of the pathogen (Hartung et al., 2010). Even
though Las has delayed movement out of young flush, its population is highest in the seed coat compared to other tissues based on
our experience. Dodder also hosts a high density of Las based on
our previous experience. We used different tissues, including young
flush, in our microscopy studies and could only observe Las when
seed coats and dodder were used. Indeed, the procedure used to
observe flagella under TEM is quite challenging for unculturable
bacteria. More robust evidence for the Las movement model can
be acquired by fluorescent microscopical analysis of green fluorescent protein (GFP)-labelled Las, as previously conducted for xylem-inhibiting Xylella (Newman et al., 2003), provided Las can be
cultured or be labelled by GFP in vivo in the future.
Those unique features of the HLB pathosystem centring on
young flush suggest it represents the Achilles’ heel for Las, and
is thus the key point for HLB/ACP management. The psyllids preferentially feed and exclusively reproduce on young, newly emerged
flush shoots of citrus, and their nymphs feed and complete their
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Fig. 9 Schematic representation of ‘Candidatus Liberibacter asiaticus’ (Las) movement with phloem sap after psyllid transmission. Phase 1: After psyllid feeding and
injecting Las into the phloem of young flush, Las initially undergoes a lag phase with no or slow growth to adapt to the environmental changes. Phase 2: Las begins
multiplication inside the young flush after an undefined period. Phase 3: With the maturation of the young flush, the infected leaf transits from a sink into a source.
Meanwhile, Las begins to move from the infected leaf to other parts of the plant, such as roots, following phloem sap from source to sink.
life stages on young flush shoots. Young flush is critical for psyllids acquiring and transmitting Las (Bové, 2006; Hall et al., 2013;
Setamou et al., 2016; Sétamou and Bartels, 2015; Tomaseto et al.,
2016). The importance of controlling psyllids during flush stages
has been recognized (Hall et al., 2016). The delayed movement of
Las out of the young flush provides opportunities to develop novel
control strategies. Interestingly, traditional citrus growers in China
not only rely on high-frequency psyllid control of young flush using
insecticides, but also remove flush (e.g. physical removal or spray
with young flush killer) during summer or winter (for certain areas).
This flush removal practice probably contributes to reducing the
ACP population, preventing ACP from acquiring Las from young
flush of HLB-positive trees and transmitting Las to young flush. The
delayed movement of Las in young flush also helps the development of targeted early detection of Las after psyllid transmission
and before HLB symptom appearance (Pandey and Wang, 2019).
In addition, targeted treatment of young flush with bactericides
might prevent Las from establishing in planta after psyllid transmission. The minimum concentration of oxytetracycline required to
suppress Las populations in planta has been suggested to be 0.68
and 0.86 µg/g under greenhouse and field conditions, respectively
(Li et al., 2019). Depending on application method, oxytetracycline
concentrations in leaf tissues have been reported to range from
0 to 0.53 µg/g (Li et al., 2019; Vincent et al., 2019), below the
concentrations required to suppress Las. It seems that successful
prevention of Las establishment after psyllid transmission requires
optimized delivery methods to increase bactericidal concentration
to the minimum concentration of bactericides required to suppress
Las populations in planta. The bactericidal effect needs to cover the
whole flush period until leaf maturation. Additionally, it is interesting to know whether the targeted treatment effect can be obtained
with non-antibiotic bactericides such as generally recognized as
safe (GRAS) antimicrobial compounds.
In summary, the flagellar characteristics of Las support a delayed movement of Las from young flush after psyllid transmission. The movement pattern of Las provides unique opportunities
for early diagnosis of HLB and targeted treatment of young flush
to control HLB.
EXP ERIMENTAL P ROC EDURES
Bacterial strains, plasmids and growth conditions
The plasmids, oligonucleotides and bacterial strains used
in this study are listed in Tables S1 and S2. Escherichia coli
cells were grown at 37 °C in Luria–Bertani (LB) medium. The
A. tumefaciens strain C58 and mutant strains were grown in
LB medium at 28 °C. Plasmids were introduced into E. coli by
heat-shock at 42 °C and into A. tumefaciens by electroporation.
Agrobacterium tumefaciens deletion mutants were generated
using the suicide vectors pNPTS138 as described elsewhere
(Hibbing and Fuqua, 2011). Cells of L. crescens strain BT-1 were
grown at 28 °C at 150 rpm in BM7 medium. Antibiotics were
used at the following concentrations: kanamycin 50 mg/mL,
ampicillin 100 mg/mL, gentamicin 10 mg/mL.
Deletion of flagellar genes and construction of
complemented strains of A. tumefaciens
DNA manipulations and PCR were performed according to standard procedures (Sambrook et al., 2006). To construct the flaA, flaD
and flgJ deletion mutants, approximately 1 kb of the upstream
and downstream regions of those flagellar genes were amplified
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by PCR from A. tumefaciens genomic DNA (oligonucleotides are
listed in Table S2), and the two fragments were ligated to produce an in-frame deletion. The approximately 2 kb fragments that
resulted were then cloned into the HindIII site of the pNPTS138
suicide vector, generating the plasmids pNPTS-FlaA, pNPTS-FlaD
and pNPTS-FlgJ (Andrade and Wang, 2019; Andrade et al., 2014).
These constructs were introduced into A. tumefaciens cells by
electroporation, and the wild-type copies were replaced by the
deleted version after two recombination events. For the first and
second recombination events, A. tumefaciens cells were selected
in LB medium without NaCl (LBON) with kanamycin and in LBON
plus 5% sucrose, respectively. The single mutants ∆flaA, ∆flaD and
∆flgJ and the double mutant ∆flaAD were confirmed by PCR. To
produce the flagellin null mutant, two fragments of 1 kb of the
upstream and downstream regions of flaA and flaC were amplified
and ligated. A fragment of 2 kb was generated and cloned into the
HindIII site of pNPTS138. This resulting construct pNTPS-FlaABC,
carrying in-frame deletion of flaA-flaB-flaC, was introduced into
A. tumefaciens flaD mutant strain by electroporation. After selections in appropriate media, the null mutant ∆flaABCD was confirmed by PCR. To complement ∆flgJ, ∆flaA, the double mutant
∆flaAD and the null mutant strains, fragments including the coding
region of flaA or flgJ genes were amplified by PCR from Las genomic
DNA. Also, the coding region of flgJ was amplified from A. tumefaciens genome. Those three fragments were inserted into the BamHI
site of the pTF53 vector that contains the constitutive promoter
Trp (Andrade and Wang, 2019; De Feyter et al., 1993), creating the
plasmids pF53-Las_FlaA, pF53-Las_FlgJ and pF53-Atu_FlgJ. The
construct pF53-FlaA_Las was used to transform ∆flaA, ∆flaAD and
∆flaABCD mutant strains. ∆flgJ mutant strain was transformed with
pF53-Las_FlgJ or pF53-Atu_FlgJ constructs. After electroporation,
the transformed A. tumefaciens cells were selected on LB medium
with gentamicin. All constructs were sequenced for confirmation.
Motility assay
The swimming motility of A. tumefaciens was assayed on semisolid agar as described previously (Merritt et al., 2007). Briefly,
bacteria cells grown in LB media for 48 h at 28 °C were stabbed
in 0.3% (w/v) agar ATGN modified medium plates (20 mM NaCl,
10 mM (NH4)2SO4, 5 mM MgSO4, 1 mM CaCl2, 0.16 mM KH2PO4,
0.32 mM K2HPO4, 0.01 mM FeSO4, 10 mM fructose, 10 mM sucrose, 0.03% casamino acid). Motility was evaluated and photographed after 3 days of incubation at 28 °C. The means of
three replicates and standard deviation values were calculated.
Statistical analysis was conducted by Student’s t-test (P < 0.05).
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medium plus specific antibiotics (Table S1) at 37 °C and 200 rpm.
Then, precultured cells were diluted 1:100 in 100 mL of LB medium
and grown at 37 °C until OD600nm = 0.6. The expression of the recombinant proteins was induced by addition of 1 mM IPTG to the
growth medium and the induction process occurred at 30 °C and
200 rpm for 6 h. After induction, E. coli cells were pelleted by centrifugation at 2348 g and 4 °C for 10 min. Pelleted E. coli cells expressing GST-FlgJLas protein were washed in phosphate-buffered
saline (PBS, pH 7.4) and suspended into lysis buffer (PBS, 100 µg/mL
lysozyme and 40 µg/mL DNAse I) and sonicated to generate
the cell lysates. After centrifugation, the cell lysates were incubated with 0.3 mL of glutathione sepharose high-performance
beads for 1 h then beads mixed with cell lysates were loaded
on a 10 mL column, according to the manufacturer’s instructions
(GE Healthcare, Pittsburgh, PA, USA). The beads were washed
four times with PBS to remove the unbound proteins and incubated with E. coli cell lysates containing 6×HisEGFP, 6×HisFLgB
or 6×HisFliE for 2 h at 4 °C. After washing three times, the proteins were eluted with PBS plus reduced glutathione (7 mg/mL).
Eluted samples were mixed with 4× SDS-PAGE loading buffer and
boiled for 5 min. The controls (Inputs) and eluted samples were
subjected to SDS 12% polyacrylamide gel electrophoresis and immunoblotting by using anti-GST (1:5000) and anti-6×His (1:3000)
(Sigma, St Louis, MO, USA) antibodies followed by secondary antibodies raised against rabbit (1:10 000) (Sigma).
Las-infected psyllids and plants
Las-infected psyllid colonies were reared in laboratory cultures
maintained on Las-infected sweet orange trees at the University
of Florida, Citrus Research and Education Center (CREC-UF, Lake
Alfred, FL, USA). Psyllid colonies were maintained in insect-proof
cages inside environmentally controlled growing chambers with
a 12-h photoperiod. Las-infected seeds were obtained from
lopsided fruits from HLB-positive grapefruit (Citrus paradisi).
Las-infected dodder plants (Cuscuta sp.) on HLB-positive periwinkle (Catharanthus roseus) were used for TEM assay. Plants
were grown in a greenhouse with a 12-h photoperiod and controlled temperature. Las-positive dodder tendrils were a gift
from Dr Ed Etxeberria (University of Florida, CREC, Lake Alfred,
FL, USA). Las in the insects, citrus plants, seed coats and dodder
plants was verified by qPCR (Hartung et al., 2010; Hilf et al., 2013;
Yan et al., 2013). A sleeve net (80 mesh) was used to cage Laspositive psyllids on young flush only. The caged plants were kept
within a bigger cage to prevent interference from other psyllids.
Isolation of Las from psyllid guts and plant materials
Pull-down assay
The constructs used in the pull-down assay are listed in Table S1.
Those constructs were used to transform E. coli strain BL21(DE3).
The transformants were precultured overnight in 3 mL of LB
About 80 Las-infected adult psyllids (Ct value = 26 to 32) were
collected from the psyllid room (CREC-UF) and used to dissect
midguts in sterile PBS under a dissecting stereomicroscope by
using depressed glass wells and fine entomological needles. After
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tissue samples were dissected, the tissues were washed three
times with PBS and transferred to a sterile Eppendorf tube containing 0.3% bacitracin in PBS (Ghanim et al., 2016). Samples
were pipetted up and down slowly to release the bacteria cells
attached to the midgut.
Selected seeds isolated from infected and symptomatic citrus plants (Ct value about 27 to 29) were washed five times in
sterile water, dried in filter paper and the coats were removed by
using high-precision straight fine tweezers. Seed coats were divided into very small pieces and suspended with 0.3% bacitracin
(Thermo Fisher, Waltham, MA, USA) in PBS.
Individual dodder tendrils positive for Las (Ct = 24 to 26) were
removed intact from parasitized plants. A segment of 10 mm was
removed from the centre of dodder stem pieces, washed five
times in sterile water, dried in paper filter, transferred to 0.3%
bacitracin in PBS and cut into very small pieces with razor blades.
The mixtures were pipetted up and down slowly to release the
bacteria. Solution containing Las was transferred to a sterile
Eppendorf tube.
Transmission electron microscopy and
antibody-labelled Las cells
Bacterial cultures of L. crescens were grown at 28 ºC in BM7 medium with 5 mM putrescine (Sigma) to OD600nm of 0.5. One droplet
of suspended bacteria was placed on formvar- and carbon-coated
400-mesh copper grids and allowed to settle for 1 min. The excess
water was wicked away with filter paper. The samples were then
stained, before drying, with 1% aqueous ammonium molybdate
(Sigma) to which a few drops of bacitracin water (20 μg of bacitracin dissolved in 10 mL water) was added as a spreading agent.
The stains were added to the grids and wicked away with filter
paper immediately. The grids were allowed to dry for 1 h before
being viewed and photographed on a Morgagni 268 transmission electron microscope (FEI Company, Eindhoven, Netherlands).
Similar procedures were repeated for bacterial suspension diluted
1:5 in PBS obtained from Las-infected psyllid midguts, grapefruit
seed coats and dodder stems.
The labelling of Las bacteria absorbed in the grids was done
as follows. Grids with absorbed Las cells were incubated with
polyclonal antibody against Las anti-OmpA raised in rabbit
(Abcam, Cambridge, MA, USA). Rabbit antisera against Las
OmpA was diluted 1:250 in PBST buffer [1x PBS, 1% (w/v) bovine serum albumen and 1% (v/v) Triton X-100]. After 1 h incubation, the grids were washed three times for 5 min each in PBST
buffer. Grids were incubated with anti-rabbit IgG (whole molecule)-gold (Sigma) diluted 1:5 in PBST buffer and incubated for
30 min. After incubation, the grids were washed three times in
PBST buffer and twice in sterile water. Finally, grids were stained
with 1% aqueous ammonium molybdate and after drying for 1 h
they were visualized and photographed on a Morgagni 268 TEM
(FEI Company).
Flagellin antibody and detection of flagellin via
western blotting
To detect the flagellin protein in different samples, the peptide
DRVSSGLRVSDAAD of Las flagellin was synthesized as an antigen to
produce Las flagellin antibody from rabbits. The total proteins were
isolated from A. tumefaciens strain GV2260, Las-infected citrus and
Las-infected psyllids. Agrobacterium tumefaciens cells were grown in
YEP medium to OD600 nm = 0.6. Cells were pelleted by centrifugation,
resuspended in 2× SDS-PAGE loading buffer and boiled for 5 min.
Protein extracts from both Las-infected and Las-free psyllids (ten insects) and citrus plants (pieces of petioles) were obtained by snap
freezing the samples in liquid nitrogen following maceration of the
tissues in tissue Lyser (Qiagen, Germantown, MD, USA). Macerated
psyllid samples were resuspended in I-PER Insect Cell protein extraction reagent (Thermo Fisher), and macerated citrus samples were
resuspended in CelLytic P Cell lysis reagent (Sigma). Resuspended
samples were centrifuged at 13 523 g for 15 min at 4 °C, then the
supernatants were diluted in 2× SDS-PAGE loading buffer. Ten microlitres of protein of each sample was loaded for 10% SDS-PAGE
electrophoresis and western blot analysis. Full-length flagellin fused
with a maltose-binding protein (MBP) tag was used as a positive
control. Las-free citrus and psyllids, and free MBP protein were used
as negative controls.
RT-qPCR analysis of Las flagellar genes
Comparative analysis of expression of the selected flagellum genes
encoded by the Las genome was verified in Las-infected plants of
Valencia sweet orange (C. sinensis) and in Las-infected psyllids as
described previously (Yan et al., 2013). All RT-qPCRs were performed
using a 7500 Fast Real-time PCR system (Applied Biosystems, Foster
City, CA, USA) with a QuantiTect SYBR Green RT-PCR kit (Qiagen). The
primers were designed from the sequence of the Las genome using
IDT SciTolls (www.idtdna.com/pages/scitools). The total reaction volume of one-step RT-qPCR was 25 μL and contained 2 × QuantiTect
SYBR Green RT-PCR Master Mix (12.5 μL), 10 μM gene-specific primers (1.25 μL), QuantiTect RT Mix (0.5 μL) and 50 ng of RNA template
(1 μL). 16S rRNA was used as the endogenous control. Reactions
were incubated at 50 °C for 30 min and at 95 °C for 15 min, then
cycled (40 times) at 94 °C for 15 s, 54–56 °C for 30 s and 72 °C for
30 s. Melting curve analysis was conducted to verify the specificity of
the RT-qPCR products. Two technical replicates and three biological
replicates were used for each of the genes. The relative fold change
in target gene expression was calculated using the formula 2−ΔΔCt
(Livak and Schmittgen, 2001). Statistical analysis of all data was conducted by Student’s t-test.
AC KNOWLEDGEMENTS
This work has been supported by Florida Citrus Initiative Program,
Citrus Research and Development Foundation, and the USDA MAC
M o l e c u l a r Plant Pathology (2 0 2 0 ) 2 1(1 ) , 109– 123 © 2019 TH E A U TH O RS. MOLECULAR PLANT PATHOLOGY PU BLI SH E D BY
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Las flagella and movement
program. We thank Dr Ed Etxeberria for the Las-positive dodder materials. This project was also supported by the National Key Research
and Development Program of China (2018YFD0201500) to T.S.Y.
DATA AVAI LABILITY S TATE M E NT
The data that support the findings of this study are available from
the corresponding author on reasonable request.
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SUP P ORTING INFORMATION
Additional supporting information may be found in the online version of this article at the publisher’s web site:
Fig. S1 Sequence alignment of the peptidoglycan hydrolase encoded by ‘Candidatus Liberibacter’ species was generated by
ClustalW. The signal peptide sequence found by PrediSi is highlighted in yellow. The SLT domain is indicated in green. CLas, ‘Ca.
Liberibacter asiaticus’ (CLIBASIA_00965); CLaf, ‘Ca. Liberibacter
africanus’ (G293_01215); CLam, ‘Ca. Liberibacter americanus’
(Lam_376); CLso, ‘Ca. Liberibacter solanacearum’ (CKC_02595);
Lcc, Liberibacter crescens BT-1 (B488_10490).
Fig. S2 Swimming motility assay of the Agrobacterium tumefaciens wild-type, ΔflaAD and ΔflaABCD mutant strains, and the
mutant strains complemented with ‘Candidatus Liberibacter asiaticus’ (Las) flaA. The mean values ± the standard deviations (n = 3)
are plotted. Mean values were compared to the wild-type, * indicate statistically significant difference (P < 0.05, Student t test).
WT, A. tumefaciens wild-type strain carrying the empty vector.
ΔflaAD and ΔflaABCD, A. tumefaciens mutant strains carrying the
empty vector. ΔflaAD + flaALas and ΔflaABCD + flaALas, mutant
strains carrying the Las flaA gene.
Table S1 Bacterial strains and plasmids used in this study.
Table S2 Oligonucleotides used in this study.
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