Jou nal o Expe imen al Bo any, Vol. 00, No. 0 pp. 1–11, 2025
h ps://doi.o g/10.1093/jxb/e a 197 Ad ance Access Publica ion 13 May 2025
TECHNICAL INNOVATION
Quan i a i e assessmen o ho mogonia induc ion in Nos oc
punc i o me by a luo escen epo e s ain
Anna Neubaue
1,2,†,
, Maca ena Inies a-Palla és
3,†,
, Consolación Ál a ez
3,
, Au élien Bailly
2,4,
,
Pé e Szö ényi
1,2,
*
,
, and Vicen e Ma iscal
3,
*
,
1
Depa men o Sys ema ic and E olu iona y Bo any, Uni e si y o Zu ich, 8008 Zu ich, Swi ze land
2
Zu ich-Basel Plan Science Cen e , ETH Zu ich, 8092 Zu ich, Swi ze land
3
Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa, Amé ico
Vespucio 49, 41092 Se illa, Spain
4
Depa men o Plan and Mic obial Biology, Uni e si y o Zu ich, 8008 Zu ich, Swi ze land
†
These au ho s con ibu ed equally o his wo k.
* Co espondence: [email p o ec ed] o [email p o ec ed]
Recei ed 6 Janua y 2025; Edi o ial decision 11 May 2025; Accep ed 11 May 2025
Edi o : Oswaldo Valdés-López, Uni e sidad Nacional Au onoma de Mexico, Mexico
Abs ac
While symbio ic plan –cyanobac e ia in e ac ions hold signi ican po en ial o e olu ionizing ag icul u al p ac ices by
educing he applica ion o a i icial ni ogen e ilize s, he gene ic unde pinnings o he symbio ic in e ac ion be ween
he plan hos and he cyanobion emain poo ly unde s ood. In pa icula , he molecula mechanisms h ough which
hos plan s induce he o ma ion o mo ile cyanobac e ial ilamen s (ho mogonia), essen ial o coloniza ion and ini i-
a ion o symbiosis, a e no well cha ac e ized. In his s udy, we p esen a no el ye objec i e me hod o quan i ying
ho mogonia induc ion, add essing limi a ions o adi ional quali a i e app oaches. We ha e de eloped a epo e
s ain o Nos oc punc i o me PCC 73102 capable o quan i ying ho mogonia induc ion in esponse o di e se bio ic
and abio ic s imuli. This epo e s ain, gene a ed ia ipa en al ma ing conjuga ion ans o ma ion, con ains he p o-
mo e sequence o p epilin pilA used o a g een luo escen p o ein (GFP) and enables quan i a i e and high- h ough-
pu moni o ing o ho mogonia induc ion using a mic opla e eade . Ou inno a i e app oach, employing a
cyanobac e ial ho mogonia epo e s ain, allows high- h oughpu sc eening o he ho mogonia-inducing e ec o
a wide a ay o en i onmen al and plan signals. This me hod is expec ed o g ea ly ad ance ou unde s anding o
he gene ic de e minan s unde pinning plan –cyanobac e ia symbioses.
Keywo ds: An hoce os ag es is, biological ni ogen ixa ion, cyanobion , ho mogonia, Nos oc punc i o me, plan –cyanobac e ia
symbiosis.
© The Au ho (s) 2025. Published by Ox o d Uni e si y P ess on behal o he Socie y o Expe imen al Biology.
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In oduc ion
Nos ocales cyanobac e ia a e pho o ophic soil bac e ia capable
o es ablishing acul a i e, diazo ophic symbiosis wi h a wide
a ie y o euka yo ic o ganisms, including dia oms, ungi,
and plan s (Nilsson e al., 2002; Adams e al., 2006; Ushe
e al., 2007; Ál a ez e al., 2023). The mos ex ensi ely s udied
cyanobac e ial symbion (cyanobion ) is Nos oc punc i o me
(Meeks e al., 2001; Meeks, 2003). Nos oc punc i o me PCC
73102 (also known as ATCC 29133) se es as a model o gan-
ism o plan –cyanobac e ia symbioses due o i s abili y o o m
endophy ic associa ion wi h isola ed species ac oss all majo
axonomic g oups o land plan s (Bonne and Sil es e ,
1981; Be gman e al., 1996; Cos a e al., 2004; Ál a ez e al.,
2020; F angedakis e al., 2023).
The es ablishmen o endosymbiosis be ween plan s and
N. punc i o me can be b oadly di ided in o h ee phases
(Johansson and Be gman, 1992; Meeks, 2003; Al a enga
e al., 2022). In he ini ial phase, he hos plan sec e es
ho mogonia-inducing ac o s (HIFs), p omp ing he di e en-
ia ion o ege a i e Nos oc cells in o mo ile ho mogonia ila-
men s (Ende lin and Meeks, 1983; Knigh and Adams, 1996;
Nilsson e al., 2006). Du ing he subsequen phase, he plan
hos is colonized by mo ile ho mogonia which a e guided by
chemoa ac an s p oduced by he hos plan . S udies in
O yza sa i a ha e demons a ed ha du ing coloniza ion,
co e componen s o he common symbiosis signaling pa hway
(CSSP; also known as common symbiosis pa hway) a e ac i-
a ed in he plan hos (Ál a ez e al., 2022). This pa hway is
essen ial o es ablishing symbio ic in e ac ions no only wi h
a buscula myco hizal ungi bu also wi h nodule- o ming
bac e ia (Pa niske, 2000; Delaux e al., 2014; Li e al., 2020).
Ul ima ely, a s able symbiosis is es ablished as he plan en aps
he cyanobion and enhances i s ni ogen ixa ion a e by in-
c easing he he e ocys equency wi hin he plan issue
(S einbe g and Meeks, 1991; Cohen and Meeks, 1997;
Meeks and Elhai, 2002; Al a enga e al., 2022). Nos oc punc i-
o me is able o di e en ia e in o h ee specialized cell ypes:
he e ocys s, ho mogonia, and akine es (Rippka e al., 1979;
S unecký e al., 2023). While he e ocys s and akine es (do -
man cells) unc ion in ni ogen ixa ion and su i al unde ha sh
en i onmen al condi ions, espec i ely, ho mogonia a e essen-
ial o he cyanobion o es ablish symbiosis wi h he hos plan
and hus a e e e ed o as he ‘plan in ec ion uni s’ (Be gman
e al., 2007). Ho mogonia di e en ia e om he ege a i e ila-
men and can be iden i ied by hei small cell size, he absence o
he e ocys s, he p esence o a ow-like cells in he poles, and—in
ce ain axa— he p esence o gas esicles (He dman and Rippka,
1988; Meeks and Elhai, 2002). Addi ionally, ho mogonia ex-
hibi pilus-like appendages on hei cell su ace and a e co e ed
in mucilage, which acili a es mo emen and hos ecogni ion
(Duggan e al., 2007; Khaya an e al., 2015).
The di e en ia ion o ege a i e cells in o ho mogonia in-
ol es he ac i a ion o >1800 genes and can be induced by
speci ic wa eleng hs o ligh , nu ien s ess, o phy ochemicals
(Rasmussen, 1994; Rai e al., 2000; Schüßle , 2000; Nilsson
e al., 2006; Campbell e al., 2007, 2008; Ha wood and
Risse , 2021). Upon s imula ion, 90–100% o cells can di e -
en ia e in o ho mogonia wi hin 18–24 h (He dman and
Rippka, 1988), wi h a li e span a e aging 72–96 h (Campbell
e al., 1998). S imuli such as symbio ic plan hos s, exposu e
o g een ligh , and he addi ion o suc ose o suc alose can sup-
p ess ho mogonia o ma ion (He dman and Rippka, 1988;
Campbell e al., 1997; Spli and Risse , 2016).
While ansc ip ional egula ion o ho mogonia di e en i-
a ion has been ex ensi ely in es iga ed, he bio ic and abio ic
ac o s ac i a ing ho mogonia o ma ion in N. punc i o me a e
poo ly unde s ood (Campbell e al., 2007, 2008; Ha wood and
Risse , 2021; Ál a ez e al., 2023). Rema kably, despi e decades
o esea ch, he HIFs sec e ed by he hos plan emain la gely
uncha ac e ized (Nilsson e al., 2002; Duggan e al., 2013;
Hashidoko e al., 2019). A comp ehensi e unde s anding o
he chemical composi ion o HIFs is pi o al o deciphe ing he
communica ion be ween he plan hos and he cyanobion du -
ing he ini ial phase o he symbio ic in e ac ion (Ál a ez e al.,
2022).
The ecen ad en o gene ically ac able model hos sys ems
makes i possible o ca y ou la ge-scale o wa d gene ic expe i-
men s, acili a ing he in e ence o he molecula mechanisms
unde pinning plan –cyanobac e ia symbio ic in e ac ions
(Wa shan e al., 2018; Cassie -Chau a e al., 2021). These e -
o s aim o shed ligh speci ically on he molecula unde pin-
nings o HIF p oduc ion and i s in luence on he in ec ion
uni o Nos oc spp., namely he mo ile ho mogonia. Howe e ,
such expe imen s demand a me hodology ha suppo s swi ,
au oma ed, and quan i a i e sc eening o ho mogonia o ma-
ion. Cu en ly, ho mogonia induc ion is e alua ed p edomin-
an ly quali a i ely using pla e mobili y assays (Liaime e al.,
2011; Spli and Risse , 2016; Nishizuka and Hashidoko,
2018; Hashidoko e al., 2019). Mo e labo -in ensi e quan i a i e
assays employ ligh mic oscopy and manual coun ing (Campbell
and Meeks, 1989; Knigh and Adams, 1996) o , al e na i ely, use
e e se ansc ip ion eal- ime PCR (RT–PCR) o quan i y he
exp ession o genes in ol ed in ho mogonia o ma ion (Liaime
e al., 2011). Howe e , none o he exis ing echniques o e s he
capabili y o apid sc eening ac oss a as a ay o samples in a
quan i a i e manne .
In his s udy, we p esen a no el app oach employing cyano-
bac e ial ho mogonia epo e s ains, acili a ing he quan i a-
i e assessmen o ho mogonia o ma ion in N. punc i o me.
Ou me hodology suppo s high- h oughpu sc eening ac oss
many di e en ex e nal condi ions and plan exuda es ha
po en ially induce ho mogonia. Consequen ly, his app oach
holds signi ican po en ial o enhance ou unde s anding o
he gene ic unde pinnings o plan –cyanobac e ia symbio ic
in e ac ions.
2| Neubaue e al.
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Ma e ials and me hods
Cyanobac e ial cul u e condi ions
The labo a o y model s ain N. punc i o me PCC 73102 (also known as
ATCC 29133 and UCD 154) was ob ained om he cul u e se ice o
he Ins i u e o Plan Biochemis y and Pho osyn hesis, Se illa, Spain. I
was ou inely main ained in solid BG11 (Rippka e al., 1979) medium
con aining 1% w/ Bac o Aga (Bec on, Dickinson and Company,
F ance). Solid cul u es we e kep on a shel a 25 °C unde educed illu-
mina ion (20–25 µmol m
−2
s
−1
) in a g ow h chambe . Liquid cul u es
we e p epa ed in BG11 om solid cul u es and incuba ed a 20–25 °C,
wi h a con inuous ligh exposu e o 45–55 µmol m
−2
s
−1
on an o bi al
shake (100–120 pm). Mu an s ains de i ed om N. punc i o me we e
main ained unde he same condi ions, bu g ow h media we e supple-
men ed wi h 25 µg ml
–1
neomycin o p ese e he gene ic modi ica ion.
Plan cul u e
Thallus issue o he ho nwo An hoce os ag es is Bonn isola e (Szö ényi
e al., 2015) was cul i a ed in liquid BCD medium (Co e e al., 1996)
unde he same condi ions as desc ibed abo e. To induce he sec e ion
o he HIF, 1 g (FW) o hallus issue was ans e ed o BG11
0
(BG11
wi hou ni ogen; Rippka e al., 1979) and supplemen ed wi h 5 mM
MES (Me ck KGaA, Ge many) o s abilize he pH a 7.4. The cul u es
we e g own in 500 ml E lenmeye lasks con aining 200 ml o BG11
0
sealed wi h a sponge plug o 1 week.
Gunne a inc o ia plan s we e cul i a ed in a g eenhouse in po s. Gunne a
mucilage con aining he HIF was ob ained om he eme ging lea es, us-
ing a s e ile spoon.
Rice (O yza sa i a) and whea (T i icum aes i um) plan s we e ge mi-
na ed axenically and g own hyd oponically in BG11. To ob ain HIF,
hey we e main ained o 1 week in BG11
0
unde illumina ion. This
BG11
0
medium was used in he expe imen s o induce ho mogonia.
Cons uc ion o N. punc i o me epo e s ains
Repo e s ains we e c ea ed by in oducing a gene ic usion casse e
in o he N. punc i o me genome ca ying he pu a i e p omo e sequences
o he N. punc i o me genes pilT2 and pilA used o a g een luo escen
p o ein (GFP; Supplemen a y Fig. S1). Using he genomic sequence o
N. punc i o me (GCA_000020025.1, (Fujisawa e al., 2017), ∼700 bp up-
s eam o he p edic ed ansc ip ion s a o pilT2 and pilA we e ampli ied
wi h he p ime s lis ed in Supplemen a y Table S1. Two plasmids we e c e-
a ed by cloning he pu a i e p omo e sequences in o pCSAM135 (Flo es
e al., 2007) in Esche ichia coli DH5α (Supplemen a y Table S2). The plasmid
pCSAM135 bea s he GFP-mu 2 gene and ep esen s a common chassis ec-
o o GFP ansc ip ional usions in cyanobac e ia. The absence o mu a-
ions in he ampli ied genomic egions was e i ied by Sange sequencing
a Eu o ins Genomics Sequencing GmbH, Ge many. The cons uc s
we e subsequen ly cloned in o he conjuga i e plasmid pRL424 (Elhai
and Wolk, 1988), p oducing he plasmids pAN2 and pMIP22 bea ing he
p
pilA
–GFP and p
pilT2
–GFP usions, espec i ely (Supplemen a y Table S2).
The plasmids pAN2 and pMIP22 we e me hyla ed in E. coli HB101 con-
aining he conjuga ion-enabling plasmid pRL623 (Elhai e al., 1997).
The plasmids we e in oduced in o he N. punc i o me genome by hom-
ologous ecombina ion using he ipa en al conjuga ion me hod (Elhai
and Wolk, 1988). The ipa en al ma ing included E. coli HB101 bea ing
he me hyla ed a ge conjugal plasmids, E. coli bea ing he helpe plasmid
pRL443 (Elhai and Wolk, 1988), and N. punc i o me. The incuba ed bac e ial
mix was subsequen ly pla ed [BG11 medium, 5% LB, 1% w/ Bac o Aga ,
opped wi h a Mixed Cellulose Fil e (0.45 µm, REF HATF08550, Me ck
Millipo e L d, USA)]. A e 24 h a 25 °C unde LED ligh ubes (45–55
µmol, 4000 K, 840), he il e s we e ans e ed o solid BG11 pla es (1%
w/ Bac o Aga ). The selec ion s a ed 48 h pos - ans o ma ion by
ans e ing he il e o BG11 pla es supplemen ed wi h 25 µM neomycin
(1% w/ Bac o Aga ). This ans e on o selec ion pla es was epea ed e e y
3 d o 2 weeks un il he appea ance o colonies. The exconjugan s we e
picked and s eaked on selec ion pla es un il axenic cul u es o he ans-
o med Nos oc s ains we e ob ained. In eg a ion o he exogenous DNA
and ull seg ega ion (absence o wild- ype DNA copies) was alida ed ia
PCR using he seg ega ion p ime s (Supplemen a y Table S2; Fig. 1).
Ho mogonia induc ion
Fo he induc ion o ho mogonia, liquid cul u es o he cyanobac e ial
s ains we e p epa ed in BG11. Six days p io o ho mogonia induc ion,
he cul u e was ans e ed o an ibio ic- ee BG11
0
ha was supple-
men ed wi h 4 mM suc alose (Me ck KGaA, Ge many) o ep ess he o -
ma ion o ho mogonia (Spli and Risse , 2016). Addi ionally, he cul u e
was homogenized by passing h ough a needle (0.8×50 mm), and he
densi y was adjus ed o a Chl a concen a ion o 0.5 µg ml
–1
. The concen-
a ion o Chl a was assessed by a 1:10 dilu ion o N. punc i o me in me ha-
nol (Mackinney, 1941).
P io o ho mogonia induc ion, he cul u es we e washed wice wi h
BG11
0
. Ho mogonia induc ion was ca ied ou by exposing he epo e
s ains o ed ligh ( h ee panels o a a - ed LED module, 50–60 μmol;
G een Powe LED module HF a ed, IONC 9290004645, Koninklijke
Philips NV, he Ne he lands), o he di e en plan exuda es. As con ols,
we used cul u es in BG11
0
wi hou any addi ional ea men .
Quan i ica ion o ho mogonia
To measu e he induc ion o ho mogonia, a VARIOSKAN LUX mic o-
pla e eade was used. Fo his, 100 ml o each cul u e was quickly homo-
genized by passing h ough a needle (0.8×50 mm), and 200 μl aliquo s
we e added in o each well o he black 96-well mic opla e (Nunc,
Denma k) o measu e he GFP luo escence, u bidi y, and Chl a au o-
luo escence. We ca ied ou a leas 12 measu emen s (12 wells, echnical
eplica es) o each biological eplica e ( ou lasks o 100 ml cul u e pe
condi ion). A blank sample wi h BG11
0
o he co ec ion o au o luo -
escence and u bidi y was used in each measu emen (measu emen was
made in a leas eigh wells). Tu bidi y was measu ed a 730 nm. Chl a au-
o luo escence and GFP we e measu ed a 650–700 nm and 485–520 nm,
a e exci a ion a 650 nm and 485 nm, espec i ely. To es he e ec o
abio ic and bio ic ac o s on GFP luo escence, a one-way ANOVA in
combina ion wi h he Tukey’s hones ly signi ican di e ence (HSD)
pos -hoc es was used in R (R Co e Team, 2008).
Mic oscopy in o ma ion
Mic oscopy images we e acqui ed using a Leica TCS SP2 con ocal
mic oscope (Leica Mic osys ems, Ge many). GFP was exci ed a
488 nm. Fluo escen emission was moni o ed by collec ion ac oss win-
dows o 500–540 nm (GFP imaging) and 630–700 nm o 670–720 nm
(cyanobac e ial au o luo escence). The images we e p ocessed using he
Fiji package (Schindelin e al., 2012).
Resul s
Cons uc ion o N. punc i o me s ains exp essing g een
luo escen p o ein in ho mogonia
We aimed a c ea ing wo epo e s ains exp essing GFP in
ma u e ho mogonia. To ob ain hese gene ically modi ied
N. punc a us s ains, we designed a cons uc bea ing a an-
sc ip ional usion o he p omo e egions o pilA and pilT2
o he g p-mu 2 gene. We selec ed hese genes because hey
Quan i a i e assessmen o ho mogonia induc ion | 3
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ha e been p e iously shown o be s ongly induced speci ically
in ho mogonia (Duggan e al., 2007; Campbell e al., 2008;
Ha wood and Risse , 2021). The p omo e usions we e
inse ed in he N. punc i o me ch omosome by homologous
ecombina ion (Fig. 1), gene a ing s ains NpPilA-GFP and
NpPilT2-GFP.
Fi s ly, gi en ha a single N. punc i o me cell o en con ains
mul iple ch omosomal copies (Meeks e al., 2001), we con-
i med he absence o wild- ype copies and e i ied he co ec
genomic in eg a ion o bo h epo e lines ia PCR (Fig. 1).
Subsequen ly, we examined GFP exp ession in hese epo e
lines. To do so, we isualized GFP exp ession in ilamen s
A
B
C
D
Fig. 1. Cons uc ion o N. punc i o me epo e s ains and hei e i ica ion by PCR. (A and B) The p omo e egions o he pilA (A) and pilT2 (B) genes a e
shown in g ay, and he coding sequences in black. P ime 1 binds o he p omo e egion, p ime 2 o he GFP sequence, and p ime 3 o he gene o in e es ,
espec i ely. In a wild- ype genome, p ime s 1 and 3 will be able o anneal and ampli y he espec i e in e ening sequence s e ch. P ime 3 has no a ge
egion o bind o. In he ans o med genome, p ime s 1 and 3 will be able o anneal bu ampli ica ion will ail because he in e ening sequence is oo long.
By con as , he p ime pai 1+2 will lead o success ul ampli ica ion. Each pu a i e ans o man s ain/line is es ed wi h bo h p ime pai s (1+2 and 1+3).
The c oss e e s o a ailed PCR elonga ion due o he p oduc being oo long. (C and D) Gel images show he e i ica ion o co ec in eg a ion in o he
N. punc i o me genome. Wild- ype s ains will only show an ampli ica ion p oduc o p ime pai 1+3, whe eas s ains ca ying he p omo e –GFP inse ion will
only p oduce a band in he gel o he p ime pai combina ion 1+2. Nos oc punc i o me has mul iple genome copies pe cell. Thus, i incomple e seg ega ion
is p esen , he sample will be posi i e o bo h p ime pai combina ions, and u he selec ion ea men is needed.
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Fig. 2. Con ocal mic oscopy o N. punc i o me s ains. Filamen s om he indica ed s ains we e aken om cul u es g own in BG11
0
medium (no induced;
A) o a e incuba ion o 24 h in he p esence o An hoce os ag es is exuda es (ho mogonia induc ion; B) and isualized by con ocal mic oscopy. Me ged
(2 channels) show he o e lay o au o luo escence (magen a) and GFP luo escence (g een). Me ged (3 channels) ep esen he o e lay o magen a
au o luo escence, b igh ield, and g een GFP luo escence. Ho mogonia a e iden i ied as sho ilamen s wi h small cells. Scale ba =20 μm.
Quan i a i e assessmen o ho mogonia induc ion | 5
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g own in BG11
0
medium a e a 24 h incuba ion wi h
A. ag es is exuda es known o induce ho mogonia using a con-
ocal mic oscope (Fig. 2). Ho mogonia we e induced in he
p esence o An hoce os exuda es, as de ailed in he Ma e ials
and me hods. As expec ed, we did no de ec GFP signal in
samples ha we e no induced, and isual inspec ion con i med
ha hey con ained only ege a i e cells. Howe e , we ob-
se ed s ong GFP luo escence in ho mogonia om he s ain
NpPilA-GFP, indica i e o ho mogonia-speci ic exp ession o
he ansc ip ional usion (Fig. 2). The s ain NpPilT2-GFP ex-
hibi ed ain GFP exp ession, sugges ing low p omo e ac i i y.
This esul is consis en wi h p e ious obse a ions epo ing
s ong and weak exp ession o he PilA and PilT2 genes, e-
spec i ely, in Nos oc cells unde going ho mogonia o ma ion
(Ha wood and Risse , 2021). Consequen ly, we selec ed he
s ain NpPilA-GFP o subsequen analyses.
G een luo escen p o ein exp ession eaches a maximum
a 18–21 h pos -induc ion
We moni o ed GFP exp ession o he NpPilA-GFP s ain
unde induc i e condi ions o de e mine he ime poin a which
GFP eaches maximum exp ession (Fig. 3). A p e-cul u e g own
in he p esence o ho mogonia- ep essing suc alose was exposed
wi h An hoce os exuda es (Fig. 3) a e ho ough washing o
induce ho mogonia di e en ia ion. As a nega i e con ol, pa
o he p e-cul u e was main ained in he p esence o suc alose.
We ound ha GFP exp ession measu ed by a mic opla e
eade (see he Ma e ials and me hods) inc eased, eaching a
pla eau a 18–21 h pos -induc ion, indica ing ull ho mogonia
di e en ia ion (Fig. 3). As he exp ession signal s ayed s able
un il 30 h, we de ined 24 h pos -induc ion as he op imal
ime poin which we used in all subsequen analyses.
While suc alose ea men inhibi s, i s emo al is known o
induce massi e ho mogonia di e en ia ion (Spli and Risse ,
2016). The e o e, o sepa a e he e ec o An hoce os exuda es
om ha o suc alose emo al on ho mogonia induc ion, we
also assessed he sole e ec o suc alose emo al a e 24 h
(Fig. 4). While suc alose emo al signi ican ly inc eased he
GFP signal compa ed wi h he suc alose- ea ed con ol, con-
i ming p e ious obse a ions (Spli and Risse , 2016), expos-
u e o An hoce os exuda es led o a much mo e d ama ic
exp ession inc ease (see ea men ‘Rep essed’ in Fig. 4).
Abio ic ac o s in luencing ho mogonia o ma ion
Ho mogonia di e en ia ion in N. punc i o me can be induced
by a ious abio ic s imuli, including changes in ligh quali y
and quan i y, as well as ans e o new g ow h media
(Campbell and Meeks, 1989; Ka oh e al., 2003). To es ou
Fig. 3. Time cou se o GFP exp ession du ing ho mogonia induc ion in he NpPilA-GFP s ain. GFP luo escence in ensi y was measu ed a a ious ime
poin s pos -induc ion using a mic opla e eade in he NpPilA-GFP s ain g own wi h suc alose ( ep essed) o induced wi h A. ag es is exuda e. To co ec o
a ying numbe s o cells in he wells, GFP luo escence was no malized by chlo ophyll au o luo escence.
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epo e s ain, we induced ho mogonia in he NpPilA-GFP
s ain which was p e iously g own in BG11
0
supplemen ed
wi h suc alose by (i) ans e ing i o a new medium wi hou
suc alose, and (ii) exposing i o ed ligh . As nega i e con ols,
we also measu ed GFP exp ession in he NpPilA-GFP s ain
g own in suc alose-con aining BG11
0
.
As expec ed, we ound ha he suc alose- ea ed samples
showed weak GFP exp ession in compa ison wi h he
wild- ype s ain (Fig. 5). T ans e o a new medium wi hou
suc alose esul ed in a signi ican inc ease in GFP exp ession
compa ed wi h he ep essed samples, al hough he le els
we e much lowe han hose obse ed wi h ed ligh exposu e
(Fig. 5).
Bio ic ac o s in luencing ho mogonia di e en ia ion
Ho mogonia di e en ia ion can be signi ican ly in luenced by
bio ic ac o s, pa icula ly he p esence o plan exuda es.
P e ious s udies ha e demons a ed he ole o plan -de i ed
compounds in modula ing ho mogonia di e en ia ion, enhan-
cing ou unde s anding o cyanobac e ia–plan symbiosis
(Meeks and Elhai, 2002; Risse and Meeks, 2013).
To in es iga e he impac o speci ic plan exuda es on ho -
mogonia di e en ia ion, we ea ed N. punc i o me cul u es
wi h exuda es om A. ag es is, Gunne a inc o ia, O yza sa i a,
and T i icum aes i um. All ou plan s a e known hos s o
N. punc i o me (Neubaue e al., 2024). The con ol ea men
included cul u es main ained wi h ho mogonia- ep essing su-
c alose. Ou esul s demons a e ha exuda es om G. inc o ia
and T. aes i um induced he highes le els o GFP exp ession,
indica ing obus ho mogonia di e en ia ion. The induc ion
by A. ag es is exuda e was mode a e, while O. sa i a exuda e e-
sul ed in he lowes induc ion among he plan exuda es es ed.
None heless, all plan exuda e ea men s led o signi ican ly
highe GFP exp ession compa ed wi h he suc alose- ea ed
con ol ( ep essed) (Fig. 6).
Discussion
By in eg a ing a high- h oughpu GFP luo escence measu e-
men , we ha e de eloped a simple, ye e ec i e, me hod o
quan i ying ho mogonia o ma ion in he model cyanobion
N. punc i o me. This echnique no only acili a es apid and ac-
cu a e quan i ica ion bu also ensu es ep oducibili y and scal-
abili y o la ge-scale s udies. The use o a mic opla e eade o
measu e GFP luo escence allows o high- h oughpu analysis,
which is essen ial o sc eening mul iple samples and condi ions
simul aneously. This me hodological ad ancemen add esses
he limi a ions o p e ious echniques (Spli and Risse ,
2016) by p o iding a quick and s aigh o wa d means o assess
ho mogonia induc ion ac oss a ious expe imen al condi ions.
Fig. 4. E ec o suc alose emo al and An hoce os exuda es on GFP exp ession in he pilA epo e s ain o N. punc i o me (NpPilA-GFP) 24 h pos -
induc ion. Rep essed: NpPilA-GFP g own in BG11
0
supplemen ed wi h suc alose; subcul u e: NpPilA-GFP ans e ed o BG11
0
wi hou suc alose;
An hoce os: NpPilA-GFP ans e ed o An hoce os ag es is exuda e. GFP luo escence is no malized by chlo ophyll au o luo escence. ns, no signi ican ,
***P ≤ 0.001 (one-way ANOVA, wi h Tukey’s HSD pos -hoc es ).
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Ou me hodology o e s a quan i a i e and high- h oughpu
app oach o sc eening he e ec s o a ious en i onmen al and
plan -de i ed s imuli. T adi ional me hods o e alua ing ho -
mogonia induc ion, such as pla e mobili y assays and ligh mi-
c oscopy, a e p edominan ly quali a i e and labo -in ensi e
(Campbell and Meeks, 1989; Liaime e al., 2011). These
me hods equi e signi ican ime and manual e o , limi ing
hei applicabili y o la ge-scale s udies. O he quan i a i e ap-
p oaches, such as RT– PCR, p o ide p ecise measu emen s
bu a e also ime-consuming and equi e specialized equipmen
and expe ise (Knigh and Adams, 1996; Campbell e al., 2007).
I is impo an o no e ha all ou p ocedu es use Nos oc
g own wi h suc alose, a non-me abolizable suc ose analog, o
inhibi ho mogonia o ma ion p io o es ing he e ec o bi-
o ic o abio ic ac o s (Spli and Risse , 2016). This ea men
was necessa y because Nos oc cul u es g own wi hou suc alose
con ained highly a iable amoun s o ho mogonia, hus mak-
ing measu emen s ha dly compa able. While suc alose ea -
men p o ides a con enien way o keeping he amoun o
ho mogonia low, p e ious s udies epo ed ha suc alose e-
mo al igge s massi e o ma ion o ho mogonia (Spli and
Risse , 2016). The e o e, when suc alose- ea ed Nos oc is ex-
posed o abio ic o bio ic ac o s in media lacking suc alose,
ho mogonia induc ion is expec ed o be caused in pa by
he e ec o suc alose emo al. Consequen ly, all ou
expe imen s include a con ol by ans e ing Nos oc o suc alose-
ee medium and compa ing his wi h a cul u e exposed o add-
i ional bio ic o abio ic ac o s (Figs 4–6). In ou expe imen al se
up, ho mogonia o ma ion was much mo e s ongly induced by
he es ed abio ic and bio ic ac o s han by suc alose emo al
alone, allowing us o sepa a e hese wo e ec s. Ne e heless,
he inclusion o a con ol assessing he sole e ec o suc alose e-
mo al is essen ial o accu a ely assess he e ec o abio ic and bi-
o ic ac o s on ho mogonia o ma ion.
As shown he e, his me hodology can be applied o s udy he
a ying in luences o bio ic and abio ic ac o s on ho mogonia
di e en ia ion in N. punc i o me. Abio ic ac o s, such as ligh
quali y and nu ien a ailabili y, also play a c ucial ole in ho -
mogonia di e en ia ion. Ou esul s show ha exposu e o ed
ligh signi ican ly induced ho mogonia di e en ia ion (Fig. 5).
This is consis en wi h p e ious s udies indica ing ha speci ic
wa eleng hs o ligh can igge ho mogonia o ma ion in
cyanobac e ia (Ka oh e al., 2003; Campbell e al., 2007).
Rega ding bio ic ac o s, ou esul s show ha exuda es om
G. inc o ia and T. aes i um a e pa icula ly po en in inducing
ho mogonia di e en ia ion, as e idenced by he highes le els
o GFP exp ession (Fig. 6). This aligns wi h p e ious indings
ha plan -de i ed compounds can signi ican ly modula e
cyanobac e ial de elopmen , enhancing symbio ic in e ac ions
(Meeks and Elhai, 2002; Risse and Meeks, 2013).
Fig. 5. E ec o abio ic ac o s ( ans e o new media and exposu e o ed ligh ) on GFP exp ession in he pilA epo e s ain o N. punc i o me (NpPilA-GFP)
24 h pos -induc ion. WT: wild ype N. punc i ome (wi hou p omo e –gene usion inse ion); ep essed: NpPilA-GFP g own in BG11
0
supplemen ed wi h
suc alose; subcul u e: NpPilA-GFP ans e ed o BG11
0
wi hou suc alose; ed ligh : NpPilA-GFP g own unde ed ligh . GFP luo escence is no malized by
chlo ophyll au o luo escence. ns, no signi ican , ***P ≤ 0.001 (one-way ANOVA, wi h Tukey’s HSD pos -hoc es ).
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The mode a e induc ion obse ed wi h An hoce os exuda es
and he compa a i ely lowe induc ion by O. sa i a exuda es
sugges a spec um o ho mogonia-inducing ac i i y ac oss di -
e en plan species (Fig. 6). The educed induc ion by O. sa i a
may also be due o lowe concen a ions o bioac i e com-
pounds, o equi emen o a longe induc ion ime, e lec ing
di e ences in he iming o mechanism o i s in e ac ion wi h
cyanobac e ia. This a iabili y unde sco es he speci ici y o
cyanobac e ial esponses o dis inc phy ochemicals, e lec ing
he e olu iona y adap a ions o N. punc i o me o i s symbio ic
pa ne s (Rai e al., 2000; Nilsson e al., 2002).
Despi e he lowe induc ion by ice exuda es, all plan ea -
men s esul ed in signi ican ly highe GFP exp ession compa ed
wi h suc alose- ea ed con ols, con i ming he e ec i eness o
hese bio ic ac o s in p omo ing ho mogonia di e en ia ion.
The obus esponse o G. inc o ia and T. aes i um exuda es
could be a ibu ed o he p esence o speci ic ho mones o sec-
onda y me aboli es ha ac as s ong HIFs. The p ecise chemical
na u e o hese HIFs emains o be elucida ed, bu hei iden i i-
ca ion could p o ide aluable insigh s in o he molecula mech-
anisms unde lying plan –cyanobac e ia symbioses (Be gman
e al., 2007; Adams e al., 2013; Duggan e al., 2013). Fu u e s ud-
ies should ocus on isola ing and cha ac e izing hese compounds
o unde s and hei oles in he symbio ic signaling pa hways.
Conclusion
Al oge he , he di e en ial esponses o plan exuda es ob-
se ed in his s udy highligh he complex and speci ic in e ac-
ions be ween N. punc i o me and i s plan hos s. Unde s anding
hese in e ac ions a he molecula le el will no only ad ance
ou knowledge o cyanobac e ial symbioses bu also pa e he
way o po en ial ag icul u al applica ions, such as bio e iliza-
ion and sus ainable c op managemen .
Supplemen a y da a
The ollowing supplemen a y da a a e a ailable a JXB online.
Fig. S1. Sequence o he g een luo escen p o ein used in he epo e
s ains.
Table S1. Plasmids used in he ans o ma ion o N. punc i o me.
Table S2. P ime s used o ampli y he pu a i e p omo e sequences and
alida e he seg ega ion.
Acknowledgemen s
We hank Lau a F ías (Ins i u e o Plan Biochemis y and
Pho osyn hesis, CSIC and Uni e si y o Se ille, Se illa, Spain) o he
echnical assis ance.
Fig. 6. E ec o plan exuda es (bio ic ac o s) on GFP exp ession in he pilA epo e s ain o N. punc i o me (NpPilA-GFP) 24 h pos -induc ion. The epo e
s ain was exposed o BG11
0
medium con aining exuda es o ou plan s, he ho nwo An hoce os ag es is, ice O yza sa i a, whea T i icum aes i um, as
well as mucilage collec ed om he gian huba b Gunne a inc o ia. GFP luo escence is no malized by chlo ophyll au o luo escence. WT: GFP luo escence
o wild- ype N. punc i o me; ep essed: NpPilA-GFP g own in BG11
0
supplemen ed wi h suc alose is shown as con ol. Fo cla i y, only s a is ically signi ican
di e ences (one-way ANOVA, wi h Tukey’s HSD pos -hoc es ) o he ep essed (suc alose) con ol o he pilA epo e a e indica ed: ns, no signi ican ,
***P ≤ 0.001.
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