Nanoparticle Delivery of Antisense miR162 Inhibits Invasive Habitat Adaption of Alternanthera Philoxeroides

Abstract

Phenotypic flexibility in adaptive traits is crucial for organisms to thrive in changing environments. Alternanthera philoxeroides, native to South America, has become an invasive weed in Asia. The mechanism by which invasive capacity is achieved remains unknown. Here, it is demonstrated that miR162 plays a crucial role in submergence survival for A. philoxeroides. These results highlight that the level of miR162 significantly increases in stems from 3 to 48 h upon water submergence, and knockdown of miR162 via TRV‐based VIGS system significantly disrupts stem elongation upon water submergence, ultimately resulting in a failure of plants protruding from the water surface. Interestingly, miR162 is not up‐regulated in the noninvasive congeneric alien species Alternanthera pungens, which is also native to South America but has retained its original habitats in Asia. The presence of anaerobic responsive elements (AREs) in the promoter sequences of MIR162 from A. philoxeroides rather than A. pungens may contribute to its invasion capacity. Importantly, nanoparticle delivery of antisense RNA oligonucleotides of miR162 significantly impairs stem elongation during water submergence. Thus, our findings reveal that the achievement of specific miRNA activity can drive rapid phenotypic variation, and miR162 has the potential as a bio‐pesticide for controlling the invasive growth of A. philoxeroides.

Full text

RESEARCH ARTICLE
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Nanoparticle Delivery of Antisense miR162 Inhibits Invasive
Habitat Adaption of Alternanthera Philoxeroides
Qianqian Hu, Erfeng Kou, Xiuzhen Liao, Ruiyi Qiu, Qi Tang, Huan Zhang,* Yun Zheng,*
Ji Yang,* and Binglian Zheng*
Phenotypic flexibility in adaptive traits is crucial for organisms to thrive in
changing environments. Alternanthera philoxeroides,nativetoSouth
America, has become an invasive weed in Asia. The mechanism by which
invasive capacity is achieved remains unknown. Here, it is demonstrated that
miR162 plays a crucial role in submergence survival for A. philoxeroides.
These results highlight that the level of miR162 significantly increases in
stems from 3 to 48 h upon water submergence, and knockdown of miR162 via
TRV-based VIGS system significantly disrupts stem elongation upon water
submergence, ultimately resulting in a failure of plants protruding from the
water surface. Interestingly, miR162 is not up-regulated in the noninvasive
congeneric alien species Alternanthera pungens, which is also native to South
America but has retained its original habitats in Asia. The presence of
anaerobic responsive elements (AREs) in the promoter sequences of MIR162
from A. philoxeroides rather than A. pungens may contribute to its invasion
capacity. Importantly, nanoparticle delivery of antisense RNA oligonucleotides
of miR162 significantly impairs stem elongation during water submergence.
Thus, our findings reveal that the achievement of specific miRNA activity can
drive rapid phenotypic variation, and miR162 has the potential as a
bio-pesticide for controlling the invasive growth of A. philoxeroides.
1. Introduction
The invasive plant Alternanthera philoxeroides, commonly known
as alligator weed, has emerged as a significant ecological threat
Q. Hu, Q. Tang, B. Zheng
State Key Laboratory of Genetic Engineering
Ministry of Education Key Laboratory of Biodiversity Sciences and
Ecological Engineering
Institute of Plant Biology
School of Life Sciences
Fudan University
Shanghai 200438, China
E-mail: zhengbl@fudan.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/advs.202416747
© 2025 The Author(s). Advanced Science published by Wiley-VCH
GmbH. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
DOI: 10.1002/advs.202416747
in various environments. Native to South
America, this species has rapidly spread
to diverse regions across the globe, in-
cluding North America, Asia, and Aus-
tralia, due to its high reproductive poten-
tial and adaptability.[1]The absence of its
native predator, Agasicles hygrophila,and
its robust clonal propagation system, char-
acterized by its ability to produce ad-
ventitious roots and shoots from stem
fragments, facilitates rapid and extensive
colonization.[2]A. philoxeroides forms dense
mats on the water’s surface, leading to pro-
found ecological disruptions. These mats
block sunlight, impairing the photosyn-
thesis of submerged aquatic plants and
disrupting aquatic ecosystems. The result-
ing loss of plant biodiversity can lead
to decreased oxygen levels in the wa-
ter, negatively affecting fish and inverte-
brate populations. Additionally, the exten-
sive growth of A. philoxeroides can hinder
water flow and recreational activities, and
its persistence in various habitats under-
scores the need for effective management
strategies.[2,3]
A. philoxeroides exhibits several remarkable adaptive mecha-
nisms that contribute to its success as an invasive species. One
key adaptation is its high tolerance to aquatic habitats, allowing
E. Kou, H. Zhang
School of Agriculture and Biology
Shanghai Jiao Tong University
Shanghai 200240, China
E-mail: zhang_huan@sjtu.edu.cn
X. Liao, Y. Zheng
College of Landscape and Horticulture
YunnanAgricultural University
No. 95 Jinhei Road, Yunnan650201, China
E-mail: zhengyun@fudan.edu.cn
R. Qiu, J. Yang
Ministry of Education Key Laboratory for Biodiversity Science and Ecologi-
cal Engineering
Institute of Biodiversity Science
Fudan University
Shanghai 200438, China
E-mail: jiyang@fudan.edu.cn
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it to rapidly grow out of the water by significantly increasing
its stem internode length within a few days.[1]RNA sequencing
analyses have shown that the expression of numerous hormone-
responsive, stress-responsive, and epigenetic regulatory genes
is dynamically regulated in response to water submergence.[4,5]
Although this rapid phenotypic flexibility enables A. philoxe-
roides to adapt locally, the specific mechanisms underlying this
adaptive capacity remain largely unknown. Moreover, no specific
factors have been identified that promote its high tolerance to
aquatic environments.
miRNAs modulate gene expression by targeting specific mR-
NAs for degradation or translational repression, enabling the
plant to fine-tune its response to environmental changes.[6]
Our previous finding has identified some water submergence-
responsive miRNAs, and co-expression network analyses show
that those submergence-responsive miRNAs may target genes
involved in gibberellic acid (GA) synthesis and abscisic acid
(ABA) signaling.[5]These results indicate that miRNA molecules
are actively involved in its high tolerance to aquatic habitats in
A. philoxeroides. Despite these clues of miRNA dynamics on its
phenotypic flexibility, whether and by which specific miRNA can
contribute to the process of rapid adaptation is largely unknown.
A long-standing question in invasive biology is how the ac-
quisition of invasive competence is related to the land-to-water
environmental transition. We approached this question by re-
examining the spatiotemporal expression patterns of miRNAs
and the phenotypes resulting from the knockdown of the miRNA
pathway. Our findings demonstrate that the miRNA pathway
plays a crucial role in stem elongation under water submergence
in A. philoxeroides. Furthermore, by characterizing the rapidly re-
sponsive miRNome of both young and old stem tissues subjected
to submergence for 3 h to 4 days, we identified a specific mi-
croRNA, Aph-miR162, that was significantly upregulated in both
young and old stems from 3 to 48 h after water submergence.
As expected, the knockdown of Aph-miR162 via the TRV-based
VIGS (virus-induced gene silencing) system significantly inhib-
ited stem elongation in A. philoxeroides underwater submergence
conditions, eventually resulting in plant death. Consistent with
the noninvasive nature of Alternanthera pungens, a congeneric
alien species of A. philoxeroides, the upregulation of miR162 in re-
sponse to water submergence was not observed in A. pungens.Se-
quence analyses of the promoters of MIR162 genes uncover that
the higher presence of AREs (Anaerobic Responsive Elements) in
A. philoxeroides compared to A. pungens suggests that the induc-
tion of miR162 in response to submergence is correlated with the
invasive capacity of Alternanther. Importantly, the nanoparticle-
delivery of antisense oligonucleotides of miR162 inhibits stem
elongation of A. philoxeroides during submergence, providing a
clue for developing an RNA-based pesticide for the control of in-
vasive plants.
2. Results
2.1. Spatiotemporal miRNome Responsive to Aquatic Habitats in
A. philoxeroides
Known for its remarkable adaptability, A. philoxeroides exhibits
rapid stem elongation in response to flooding conditions, en-
abling it to elevate above the water surface and effectively mitigate
submergence stress. To explore the role of miRNAs in this stem
elongation response and their potential to regulate the plant’s
thriving growth in aquatic habitats, we extracted total RNA from
young stems (including internodes 1 and 2) and old stems (in-
cluding internodes 3 and 4) subjected to varying durations of wa-
ter submergence (0, 3, 24, 48, 72, and 96 h) (Figure 1a). Subse-
quently, we conducted comprehensive miRNA sequencing anal-
yses. The miRNA-seq profiles were consistent across multiple
samples for both young stems and old stems at different time
points (Figure S1a, Supporting Information). Then, we evaluated
the global profiles of all identified miRNAs for each sample, and
the results show that the overall dynamics of miRNA changes in
both young and old stems were similar between upland and water
submergence conditions (Figure 1b, Table S1, Supporting Infor-
mation). In old stem tissues, compared to the levels observed at 0
h, A. philoxeroides showed a slight increase in miRNA levels at the
24 h mark under both upland and water submergence conditions
(Figure 1b). However, there was a significant decrease in miRNA
levels at 48 and 72 h points (Figure 1b). In contrast, the miRNA
levels in young stem tissues remained relatively stable under both
upland and water submergence conditions up to the 48 h mark
(Figure 1b). A significant decrease was observed at 72 h, but the
levels rapidly returned to normal at 96 h (Figure 1b). These analy-
ses indicate that the overall patterns of miRNA dynamics in stem
growth are comparable between upland and water submergence
conditions.
Despite the general trends indicating that both growth en-
vironments influence miRNA expression in a similar manner,
many specific miRNAs were found to be up-regulated more than
twofold in water submergence conditions compared to upland
conditions (Figure 1b). To characterize specific miRNAs that re-
spond to water submergence in A. philoxeroides, we focused on
samples collected from three to 72 h of submersion. As shown in
Figure 1c, several highly conserved miRNAs, including miR156,
miR159, miR160, miR162, miR164, miR166, miR167, miR168,
miR171, miR172, miR319, miR390, miR395, and miR396, ex-
hibited significant de-regulation under submerged conditions.
Additionally, several non-conserved miRNAs, such as miR535,
miR8005, and miR8155, were also de-regulated in response to
water submergence (Figure 1c). This suggests that specific miR-
NAs may play a crucial role in the adaptive responses of A.
philoxeroides to aquatic environments. To assess the broader sig-
nificance of certain miRNA responsiveness as a common regu-
latory mechanism during water stress and subsequent adapta-
tion, we examined the petiole elongation capacity of several Ara-
bidopsis mutants deficient in miRNA biogenesis and activity. This
phenotypic trait is crucial, as Arabidopsis plants typically elon-
gate their petioles to rise above water. The results show that com-
pared to wild type (Col-0), hyl1-2 and ago1-27 mutants, which are
known to impair miRNA processing and function, displayed sig-
nificantly reduced petiole elongation after submergence (Figure
S1b,e, Supporting Information). This finding underscores that
miRNA plays a conserved role in facilitating rapid growth above
the waterline, extending from Arabidopsis to Alternanthera.
Further investigations were conducted on the growth pheno-
types of two specific miRNA mutants: mir159abc and mir164abc,
each expected to elucidate individual miRNA contributions to
submergence adaptation. Notably, mir159abc mutant showed
a marked inhibition of petiole elongation upon submergence,
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Figure 1. miRNA profiling responding to water submergence in A. philoxeroides. a) The phenotypes of upland and water-logged A. philoxeroides at
different time points. The left side displays the overall growth phenotypes, while the right side shows the morphology of the second internode. Scale
bars are shown. b) Overall levels of miRNAs in young stems (internodes 1 & 2) and old stems (internodes 3 & 4) under different conditions. The
fold changes of miRNA levels were calculated relative to 0 h. Data are presented as mean ±standard errors (SE). c) Volcano analysis showing typical
differentially expressed miRNAs in stems at different time points after submergence. The red dots represent upregulation, while the blue dots represent
downregulation, and the gray dots indicate no change.
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suggesting its essential role in facilitating growth under these
conditions. In contrast, Col-0 and mir164abc mutant did not
exhibit this inhibitory phenotype (Figure S1c,e, Supporting
Information). Given that MYB33 and MYB65 are targets of
miR159, we further show that myb33 myb65 double mutant[7]
exhibited increased petiole elongation, contrasting with the
reduced elongation seen in mir159abc mutant (Figure S1d,e,
Supporting Information). This observation implies that the
regulation of petiole elongation by miR159 may operate through
MYB33 and MYB65, and the miRNA-mediated regulatory mech-
anisms underlying the rapid emergence of plants above the
water surface under flooding conditions may be evolutionarily
conserved from Arabidopsis to Alternanthera.
2.2. Both miRNA Biogenesis and Action Promote Stem
Elongation of A. philoxeroides in Aquatic Habitats
Both our comprehensive miRNA profiling analyses and a previ-
ously published study have consistently demonstrated that ≈100
miRNAs exhibit significant expression changes in response to
water submergence in A. philoxeroides,[5]indicating that the en-
tire pathway involving miRNA biogenesis and action may be nec-
essary for adaptation to aquatic habitats. To test this hypothesis,
we first needed to develop an efficient method to genetically in-
terfere with the activity of the miRNA pathway in A. philoxeroides.
A previous study reported that the potato virus X-based virus-
induced gene silencing (VIGS) approach is a feasible method
for knockdown of gene expression in A. philoxeroides.[8]How-
ever, the efficiency of this method in our lab, as well as in several
other labs, has been very low. This may be due to the strong re-
sistance exhibited by the invasive plant A. philoxeroides to potato
virus X-based inoculation under our growth conditions. To en-
hance the inoculation efficiency, we focused on the TRV (tobacco
rattle virus)-based VIGS system, which has proven to be the most
effective method of gene silencing in many horticultural plants
and crops.[9–11]TRV offers several distinct advantages over other
viruses developed for VIGS, including relatively mild infection
symptoms, the ability to infect large patches of neighboring cells,
and efficient migration to growing meristems, thereby reaching
new tissues throughout the plant.[12,13]Before optimizing this
method, we established a workflow to assess the aquatic habitat
adaptation ability of A. philoxeroides (Figure 2a). Initial branches
with four to five pairs of leaves were used for inoculation, fol-
lowed by culturing the inoculated branches in darkness for two
days. These branches were then split into two groups for a 10-
day growth period: one group was grown upland, while the other
was submerged in water. Finally, the length of each stem seg-
ment between nodes was measured. To determine the optimal
concentration of agrobacteria for efficient inoculation, we first
tested two positive reporters: pTRV2-GFP and pTRV2-AphPDS
(phytoene desaturase). Unlike the usual concentration of OD600 =
0.15 used for most VIGS vectors, a significantly higher concentra-
tion of OD600 =0.5 of Agrobacterium strain GV3101 was required
for the pTRV2 vector to inoculate the leaves of A. philoxeroides
effectively (Figure S2a, Supporting Information). Using this op-
timized method, we successfully expressed GFP in A. philoxe-
roides, as evidenced by the distinct fluorescence signals detected
not only within the cytoplasm of directly infiltrated leaves but
also in adjacent newly developed leaves (Figure S2b, Supporting
Information). Additionally, we successfully knocked down PDS
of A. philoxeroides, in which the leaves inoculated with pTRV2-
AphPDS, rather than the blank pTRV vector, turned yellow, and
new leaves developed a pale appearance (Figure S2c, Support-
ing Information). The expression level of AphPDS decreased to
≈30% following pTRV2-AphPDS inoculation (Figure S2d,Sup-
porting Information). These attempts demonstrated that TRV-
based VIGS can be used for gene silencing in A. philoxeroides.
To investigate whether the intact miRNA pathway is involved
in the aquatic habitat adaption of A. philoxeroides, we employed
the TRV-based VIGS system to knock down the expression of
AphDCL 1, the main miRNA biogenesis gene, and AphAGO1,
the main miRNA effector. The inoculation of TRV-AphDCL1 and
TRV-AphAGO1 into the leaves resulted in the expression of the
two target genes decreasing to ≈20% to 40% of that observed with
the blank vector inoculation (Mock control) (Figure 2b). As shown
in Figure 2c, the height of the plants significantly decreased in
both cases of knocking down either AphDCL 1 or AphAGO1. Sta-
tistical analysis of the stem internode length from the indicated
samples further confirmed that overall elongation was signifi-
cantly impaired when either AphDCL1 or AphAGO1 was knocked
down (Figure 2d). Notably, the elongation of internodes 1 and 2
was particularly affected (Figure 2d), suggesting that responsive
signals are preferentially distributed at the top. Consistently, the
impaired elongation of internodes 1 and 2 predominantly con-
tributed to the observed phenotypes in both cases of AphDCL1
and AphAGO1 knockdown (Figure 2d). These results indicate
that an intact miRNA pathway is necessary for the aquatic habitat
adaptation of A. philoxeroides.
2.3. Submergence-Induced miR162 Mediates Stem Elongation in
A. philoxeroides
To streamline the potential development of miRNA-based pesti-
cides, we hypothesized that specific miRNAs might regulate stem
elongation under flooding conditions. To test this, we analyzed
the abundance of miRNA isoforms in A. philoxeroides. Based on
this analysis, miR162 was selected for further study because it
was significantly upregulated in the stems of A. philoxeroides after
3 h of flooding treatment (Figure S3a, Supporting Information).
Additionally, miR162 is highly conserved across a wide range
of plant species, highlighting its biological significance.[14]Fur-
thermore, miR162 is encoded by only two genes in A. philoxe-
roides, which simplifies its manipulation in knockdown experi-
ments and ensures more effective suppression of its expression.
These characteristics make miR162 an ideal candidate for devel-
oping strategies to control the invasive growth of A. philoxeroides.
By carefully examining the dynamics of miR162 at different
time points in both young stems and old stems under water
submergence using RT- qPCR analyses, we show that in young
stems, miR162 levels were rapidly increased to more than twofold
at 3 h after water submergence, and this increase lasted for ≈2
days and peaked at 48 h (Figure 3a). The rapid up-regulation of
miR162 was consistent with the results of small RNA sequenc-
ing analysis (Figure S3a, Supporting Information). In contrast,
no significant changes in miR162 levels were observed in young
stems grown under upland conditions (Figure S3a, Supporting
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Figure 2. Knockdown of AphDCL1 and AphAGO1 impairs submergence-induced stem elongation in A. philoxeroides. a) Workflow for VIGS in A. philoxe-
roides. Select uniform-sized plants containing four internodes, and transplant them into soil, ensuring that two internodes are buried underground and
two internodes remain above ground. Allow the above-ground portion to grow two new internodes, typically containing eight to ten leaves. Next, inocu-
late each leaf with Agrobacterium solution containing the VIGS vectors. Keep the plants in a dark and moist environment for two days. After this period,
divide the plants into two groups: one group is submerged in water, while the other group continues to grow on upland. Finally, observe the growth
phenotypes after ten days. b) Expression of AphDCL1 and AphAGO1 in stems of VIGS-treated plants. Data are presented as mean ±standard errors
(SE) from three biological replicates. AphUBC was used as an internal control. c) Plant height comparison of water-logged A. philoxeroides after silencing
AphDCL1 and AphAGO1 with Mock. Scale bars =5 cm. d) Statistical analyses of the elongation ratio of internode after submergence treatment. Five
plants were analyzed for each group, and each dot represents one internode. ****p<0.0001, ***p<0.001, and *p<0.05, ns indicates not significant;
Student’s t-test was performed for (b) and (d).
Information). In old stems, the increase in miR162 levels oc-
curred later, at 24–48 h after water submergence, but not as
rapidly as in young stems (Figure 3a). Similarly, no up-regulation
of miR162 was seen in old stems grown under upland conditions
(Figure S3a, Supporting Information). This rapid responsiveness
of miR162 to water submergence suggests that its up-regulation
might be involved in the stem elongation of A. philoxeroides in
aquatic environments.
To determine whether the accumulation of miR162 con-
tributes to its aquatic habitat adaptation in A. philoxeroides,we
attempted to knock down miR162 levels using the TRV-based
VIGS system. In A. philoxeroides, two genes encode MIR162:
MIR162a and MIR162b. To investigate whether AphMIR162a,
AphMIR162b, or both respond to water submergence by induc-
ing the production of mature miR162, we conducted RT-qPCR
analyses to examine the levels of precursor miR162 (pri-miRNA).
The results showed that the expression of pri-miR162b, but not
pri-miR162a, was upregulated under water submergence
(Figure 3b). Subsequently, we performed knockdown experi-
ments targeting miR162 using the TRV-based VIGS system. Two
fragments, each specific to either AphMIR162a or AphMIR162b,
were separately cloned into the pTRV2 vector. These plasmids
were introduced into the leaves of A. philoxeroides, and the
efficiency of miR162 knockdown was assessed by measuring
mature miR162 levels through RT-qPCR analyses. As shown in
Figure 3c, the levels of pri-miRNA were significantly reduced in
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Figure 3. miR162 mediates submergence-induced stem elongation in A. philoxeroides. a,b) RT-qPCR analyses showing levels of miR162 (a) and pri-
miR162 (b) in stems of A. philoxeroides at different time points after water-logging. c) RT-qPCR analyses showing the levels of pre-miR162 (left) and
miR162 (right) in stems of A. philoxeroides inoculated by the pTRV2-VIGS vectors. For (a–c), data are presented as mean ±standard errors (SE) from
three biological replicates. AphU6 was used as an internal control. d) Growth phenotypes of water-logged A. philoxeroides after silencing two AphMIR162
genes. Mock was the control. Scale bars =5 cm. e) Statistical analyses of the elongation ratio of internode after submergence treatment. Five plants were
analyzed for each group, and each dot represents one internode. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05, ns indicates not significant;
Student’s t-test was performed for (a, b, c, e).
samples inoculated by either the pTRV2-AphMIR162a or pTRV2-
AphMIR162b constructs compared to the mock treatment. This
reduction indicates that the introduction of these constructs
effectively suppressed pri-miRNA levels. Correspondingly, the
levels of mature miR162 were also markedly downregulated
following the introduction of the pTRV2-AphMIR162a or
pTRV2-AphMIR162b plasmid into the leaves of A. philoxeroides
(Figure 3c). These findings are in line with the observed strong
responsiveness of MIR162b transcription to water submer-
gence, as illustrated in Figure 3b. We then compared the overall
growth phenotypes of A. philoxeroides plants inoculated with the
mock control, pTRV2-AphMIR162a, or pTRV2-AphMIR162b.
The results show that the plants inoculated with the pTRV2-
AphMIR162b plasmid exhibited a significantly reduced height
compared to the mock control (Figure 3d). In contrast, the
plants expressing pTRV2-AphMIR162a showed only a slight
reductioninheight(Figure3d). Statistical analyses of stem
length confirmed that the introduction of pTRV2-AphMIR162b
significantly inhibited the elongation of internode 1 and had
a slight inhibitory effect on the elongation of internodes 2
and 3 (Figure 3e). In contrast, the effects of knocking down
AphMIR162a were very slight (Figure 3e). This suggests that
the suppression of AphMIR162b may have a more pronounced
impact on plant growth compared to the relatively minor effect
of AphMIR162a, highlighting potential differences in their
roles in regulating plant development and stress responses. To
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determine whether the effects of miR162 on stem elongation are
due to changes in cell expansion, we measured the lengths of
surface cells on the stems. As shown in Figure S3b,c (Supporting
Information), the cell lengths in the pTRV2-AphMIR162b group
were noticeably shorter than those in the mock control.
To investigate how miR162 promotes stem elongation un-
der flooding stress, we focused on light, hormone, and cell
elongation-related genes previously reported to be upregulated
by waterlogging treatment.[15,16]We then used RT-qPCR to exam-
ine the expression of these genes following miR162 knockdown.
AsshowninFigureS3d (Supporting Information), positive reg-
ulators such as PIF7, BZR1, GID1B, and several expansin genes
(EXPA1, EXPA3, EXPA4,andEXPA5) were significantly down-
regulated, while negative regulators like GAI and RGL1 were
substantially upregulated. These coordinated changes in gene ex-
pression, spanning multiple hormonal pathways and cell wall
modification components, offer mechanistic insights into the
suppression of stem elongation following miR162 knockdown,
underscoring miR162’s pivotal role in integrating various sig-
naling pathways during the submergence response. Collectively,
these results demonstrate that miR162 plays a critical role in pro-
moting stem elongation, which is essential for the adaptation of
A. philoxeroides to aquatic habitats.
2.4. Responsiveness of miR162 to Aquatic Habitats is Specific to
Invasive Plant A. philoxeroides Rather than Noninvasive Plant
A.pungens
As previously mentioned, A. pungens is evolutionarily closely re-
lated to A. philoxeroides, with both species native to South Amer-
ica. However, upon their introduction to East Asia in the 1930s,
A. philoxeroides rapidly established itself as one of China’s top
100 invasive plants due to its robust asexual propagation and
its adaptability to various habitats. In contrast, A. pungens main-
tained its preference for upland conditions in Asia. As shown in
Figure 4a, both species grow similarly in upland environments.
However, when submerged, the stems of A. philoxeroides typically
elongate and break the water’s surface within 1–2 days, whereas
A. pungens lacks this capability and ultimately withers and dies
in such conditions (Figure 4a). To investigate whether the induc-
tion of miR162 contributes to the differences in invasive poten-
tial between A. philoxeroides and A. pungens, we conducted RT-
qPCR analyses to measure miR162 levels at various time points
before and after water submergence. As expected, A. philoxeroides
showed a significant increase in miR162 levels at 3 h after sub-
mergence, peaking at 48 h (Figure 4b), which aligns with pre-
vious observations (Figure 3a). In contrast, miR162 levels in A.
pungens remained consistently low and stable, even 72 h after
submergence (Figure 4b). This indicates that miR162 is not re-
sponsive to aquatic habitats in A. pungens.
To explore why miR162 is induced by water submergence in
A. philoxeroides but not in A. pungens, we analyzed the promoter
sequences (≈2 kb upstream of the transcription site, TSS) of Aph-
MIR162a and AphMIR162b in both species for specific cis-acting
regulatory elements using the PlantCARE database.[17]These
analyses identified that the promoter region of AphMIR162b in A.
philoxeroides contains four highly conserved Anaerobic Respon-
sive Elements (AREs, VVAAACCAVV, where V represents A, C,
or G) (Figure 4c; Figure S4, Supporting Information, highlighted
in dark blue shadow) and two degenerate AREs (AAACCA, with
possible T as the flanking nucleotides) (Figure 4c; Figure S4,
Supporting Information, highlighted in light blue shadow). In
contrast, the promoter regions of AphMIR162a in A. philoxe-
roides and both AphMIR162 genes in A. pungens were found
to either lack ARE entirely or contain only 1 to 2 degenerate
ARE elements (Figure 4c; Figure S4, Supporting Information).
AREs are recognized for their roles in mediating responses to
anaerobic conditions.[18–21]The presence of AREs suggests that
AphMIR162b may be specifically regulated by low-oxygen envi-
ronments, potentially contributing to the submergence survival
strategies. Given that anaerobic environments are a hallmark of
aquatic habitats,[22]the presence of more conserved AREs in the
promoter of AphMIR162b likely plays a critical role in the adap-
tation of A. philoxeroides to such environments.
2.5. Nanoparticle-Delivered Antisense RNA Oligonucleotides of
miR162 Inhibits Submergence-Induced Stem Elongation of A.
philoxeroides
Given that the up-regulation of miR162 is essential for stem elon-
gation in A. philoxeroides during water submergence, it allows
the plant to grow above the water surface and contributes to its
survival in aquatic habitats. Thus, there is potential to develop a
biocontrol method based on the sequestration of miR162 activ-
ity. To pursue this objective, we aimed to establish an innovative
biocontrol strategy utilizing RNA-based pesticides to manage the
invasion of A. philoxeroides. This approach could effectively dis-
rupt the growth of this invasive species, offering a targeted so-
lution for controlling its spread in aquatic environments. Previ-
ous research has demonstrated that nanoparticle-packaged RNA
oligonucleotides exhibit excellent programmability, high delivery
efficiency, and their slow-release characteristics ensure the sta-
bility of the delivered RNA.[23–26]Building upon these findings,
we investigated the application of nanoparticle-packaged RNA
oligonucleotides in A. philoxeroides. To track the localization of
these nanoparticles within A. philoxeroides, we used Cy3-labeled
tetrahedral DNA framework (TDF) to trace its transport. By in-
jecting the nanoparticles into the leaves and observing the distri-
bution of Cy3 fluorescence 12 h post-injection, we confirmed that
TDF could penetrate leaf cells (Figure 5a). Additionally, we de-
tected Cy3 fluorescence in the stem adjacent to the injected leaves
at 48 h post-injection, indicating that TDF was transported down-
ward from the leaves to the stem, fulfilling its intended delivery
function (Figure 5a). To assess the transport efficiency of TDF in
A. philoxeroides, we established different concentration gradients.
The results showed that higher concentrations (from 100 to 1000
nM) led to greater amounts of TDF being delivered and trans-
ported into the plants (Figure 5a). Based on a previous finding[27]
and considering both effectiveness and cost, a final concentration
of 200 nM miR162-as was chosen as sufficient for further delivery
and growth inhibition studies. We further utilized atomic force
microscopy (AFM) and dynamic light scattering (DLS) to ana-
lyze their size and morphology (Figure 5b,c), revealing several
3D tetra-structures with an average size of ≈5 nm. Thus, these
results indicate that the method of delivering RNA using TDF
nanoparticles is effective in A. philoxeroides.
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Figure 4. Upregulation of AphMIR162b is associated with invasion capacity in A. philoxeroides. a) Plant height comparison of upland (two left panels)
and water-logged (two right panels) A. philoxeroides with A. pungens for 20 days after submergence treatment. b) miR162 levels at different time points
in stems of A. philoxeroides and A. pungens after submergence treatment. Data are presented as mean ±standard errors (SE) from three biological
replicates. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05 were obtained using Student’s t-test. ns, not significant. c) A schematic diagram
shows the distribution of AREs (Anaerobic Responsive Elements) in the promoter sequences of MIR162 genes in invasive species A. philoxeroides and
noninvasive species A. pungens. The dark blue shading highlights the conserved ARE sequence (VVAAACCAVV), where “V” represents A, C, or G. The light
blue shading represents the degenerate ARE sequence (AAACCA), where possible T can be the flanking nucleotides. The arrows indicate the locations
of the AREs upstream of the TSS (transcription start sites).
To further validate this approach and investigate its biological
applications, we conducted experiments to assess the effects of
TDF-delivered antisense miR162 oligonucleotides on stem elon-
gation in A. philoxeroides in aquatic habitats. As illustrated in
Figure 5d, we designed and synthesized four types of TDF struc-
tures, each loaded with one, two, three, or four antisense RNA
oligonucleotides that are complementary to miR162 (miR162-as)
that were accommodated at the vertices of the TDF. We named
these structures TDF@miR162-as (1+), TDF@miR162-as (2+),
TDF@miR162-as (3+), and TDF@miR162-as (4+), respectively.
In detail, the miR162-as oligonucleotides were designed with a
poly(A) tail, allowing them to hybridize with the poly(T) sticky
ends extended from the vertices of the TDF. We characterized
the successful assembly of the TDF nanostructure and the pre-
cise loading of miR162-as using gel electrophoresis. The bands
gradually shifted upward as more miR162-as oligonucleotides hy-
bridized at the vertices (Figure S5a, Supporting Information), in-
dicating successful loading of the miR162-as. Subsequently, we
inoculated the leaves of A. philoxeroides with these TDF-loaded
antisense RNA oligonucleotides targeting miR162 and normal-
ized the miR162-as with a final concentration of 200 nM. Af-
ter 10 days of water submergence, we observed the phenotypes
related to stem elongation. The results indicated that all TDFs
carrying antisense miR162 oligonucleotides exhibited a signifi-
cant inhibitory effect on stem elongation compared to the two
negative controls: naked antisense miR162 oligonucleotides and
phosphate-buffered saline (PBS) alone (Figure 5e; Figure S5b,
Supporting Information). Notably, we found that the TDFs as-
sembled with two antisense miR162 oligonucleotides exhibited
the strongest inhibitory effects on stem elongation (Figure S5b,
Supporting Information). We attribute this effect to the con-
formation of the miR162-as on the TDF, as well as the in-
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Figure 5. Nanoparticle-mediated delivery of antisense oligonucleotides of miR162 inhibits submergence-induced stem elongation in A. philoxeroides.
a) The localization and transportation of Cy3-labelled TDF within A. philoxeroides.Scalebars=50 μm. b) AFM characterization image of TDF. Scale
bars =60 nm. c) DLS analysis of TDF. d) Illustration of the design and assembly of one, two, three, and four miR162-as onto TDF structures to
generate TDF@miR162-as (1+), TDF@miR162-as (2+), TDF@miR162-as (3+) and TDF@miR162-as (4+). e) Plant height comparison of water-logged
A. philoxeroides after delivery by TDF nanoparticle-packaged as-miR162 (TDF@miR162 (2+)) and the mock control (miR162-as with no nanoparticle).
Scale bars =5 cm. f) Statistical analyses of the elongation ratio of internode in (e). Five plants were analyzed for each group, and each dot represents
one internode. ****p<0.0001, ***p<0.001 and **p<0.01 were obtained by Student’s t-test.
creased steric hindrance encountered by miR162 when recog-
nizing TDF@miR162-as (3+) and TDF@miR162-as (4+). These
findings suggest that the utilization of nanoparticle-delivered an-
tisense miR162 oligonucleotides is a promising strategy for con-
trolling the growth of A. philoxeroides in aquatic environments.
To further validate the impact of miR162 knockdown, we em-
ployed target-directed miRNA degradation (TDMD)[28,29]to in-
vestigate the effects of nanoparticle-delivered antisense miR162
on endogenous miR162 levels in A. philoxeroides under flood-
ing. We used TDF to deliver an antisense sequence of miR162
with an additional three-nucleotide insertion (GUA) at positions
10–11, which corresponds to the well-known seed sequences of
miRNA. This modification created a simulated target, facilitating
the degradation of miR162 through the TDMD mechanism. We
then quantified miR162 levels using stem-loop RT-qPCR and ob-
served a more significant reduction in miR162 expression com-
pared to the control. Concurrently, stem elongation in the plants
was markedly inhibited (Figure S5c–e, Supporting Information).
These results demonstrate that TDF@miR162-asTDMD (2+)ef-
fectively knocks down miR162, thereby suppressing stem elon-
gation in A. philoxeroides under flooding stress. Collectively, we
conclude that inhibition of either miR162 biogenesis or activ-
ity effectively prevents stems from protruding above the water
surface. This highlights the potential for targeting miR162 as a
strategic approach to control the growth and spread of this inva-
sive species in aquatic habitats.
3. Discussion
miRNAs are pivotal regulatory molecules that play crucial roles
in various biological processes, including development and the
response to environmental stress.[6,30]In the context of A. philoxe-
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roides, the phenotypic flexibility observed in invasive aquatic habi-
tats is not attributed to changes in genetic information. Instead,
epigenetic regulation emerges as a key mechanism underpin-
ning this adaptability. In this study, we identified a specific mi-
croRNA, miR162, which plays a crucial role in the establishment
of its invasive capacity in aquatic environments. We present three
lines of evidence to suggest that inhibiting miR162 could be a
feasible strategy for the biocontrol of A. philoxeroides. First, we
demonstrate that a deficiency in the entire miRNA pathway, in-
cluding both miRNA biogenesis machinery and miRNA activity,
can effectively impede the growth of A. philoxeroides in aquatic
habitats. Second, we show that the knockdown of miR162 lev-
els using the TRV-based VIGS silencing system is sufficient to
inhibit the growth of A. philoxeroides in these environments.
Lastly, we successfully applied nanoparticle-delivered antisense
miR162-based pesticides to disrupt the growth of A. philoxeroides
in aquatic habitats. In the future, we believe that a combination
of approaches will often be more effective in controlling this in-
vasive species. For example, after a round of mechanical cutting
to reduce the bulk of the A. philoxeroides population, miRNA-
based treatments could be applied to target the remaining plants
at the molecular level. This approach would reduce competi-
tion for resources, minimize the amount of miRNA-based agents
needed, and address the underlying genetic processes that drive
regrowth. Additionally, combining miRNA regulation with tradi-
tional strategies can help mitigate some of the negative impacts
associated with any single method. For example, using a low dose
of herbicides in combination with miRNA-mediated gene silenc-
ing could reduce the environmental footprint of chemical control
while still achieving effective weed management.
In our experiments, the use of TDF nanoparticles exhibits
the ability to penetrate plant cells directly, which enhances the
stability of the antisense miR162 that they carry. As a result,
TDF nanoparticles demonstrated a significantly stronger efficacy
in sequestering miR162. Notably, when two antisense miR162
molecules were attached to the TDF nanoparticles, the effi-
ciency was higher than that observed with four antisense miR162
molecules. This phenomenon may be explained by the saturation
of the binding sites on the tetrahedral nanoparticles, which could
hinder further entry into plant cells when overloaded. Overall,
these findings indicate that the selection of delivery systems and
the optimization of RNA load are critical for the effective applica-
tion of antisense miRNAs in controlling the growth of A. philoxe-
roides and potentially other invasive species. Further exploration
of nanoparticle design and its interaction with plant cells could
lead to more effective biocontrol strategies to manage invasive
species. Given that foliar spraying is the most practical and effi-
cient method for RNA pesticide application, combining it with
other approaches, such as mechanical cutting, will be considered
for enhanced effectiveness.
Nanoparticle-delivered antisense RNA oligonucleotides target-
ing miR162 effectively inhibit stem elongation in A. philoxeroides
when exposed to aquatic habitats. This intervention suggests that
the suppression of miR162 disrupts the plant’s normal growth
responses to submerged conditions, potentially affecting its inva-
sive potential and adaptability in aquatic environments. This tar-
geted approach highlights the role of miR162 in regulating stem
elongation and could offer insights into managing the growth of
A. philoxeroides in its invasive habitats. Given that A. philoxeroides
has become an invasive species across Asia, North America, and
Australia due to its high reproductive potential and adaptability,
we believe that nanoparticle-delivered miR162 antisense oligonu-
cleotide pesticides offer a viable strategy for its global control. Ad-
ditionally, our findings may provide clues for generating crops via
molecular breeding that can tolerate flooding resilience.
4. Conclusion
The rapid spread of invasive species poses a major challenge
to biodiversity conservation. Alternanthera philoxeroides, native to
South America, has globally infiltrated various habitats due to its
strong adaptability, emerging as a significant ecological threat. A.
philoxeroides exhibits several remarkable adaptive mechanisms
that contribute to its success as an invasive species. One key
adaptation is its high tolerance to aquatic habitats, allowing it
to rapidly grow out of the water by significantly increasing its
stem internode length within a few days. Although mechani-
cal clearance and chemical pesticide spraying have shown sig-
nificant improvements in controlling Alternanthera philoxeroides,
it is essential to note that these methods come with consider-
able annual costs and environmental concerns related to chem-
ical contamination. Therefore, understanding the mechanism
by which A. philoxeroides can rapidly protrude its stem out of
the water’s surface to survive submergence is crucial, and the
development of an effective biological control strategy for con-
trolling this invasive species is urgently needed. In this study,
we identified miR162 plays a vital role in A. philoxeroides’ abil-
ity to thrive in aquatic environments and establish its invasive
capacity. We provide two lines of evidence to suggest that in-
hibiting miR162 could be a promising approach for controlling
the spread of A. philoxeroides through biological means. First,
we show that knockdown of miR162 levels using the TRV-based
VIGS silencing system is sufficient to inhibit the stem elongation
of A. philoxeroides in these environments. Second, we success-
fully applied nanoparticle-delivered antisense oligonucleotides
of miR162 to disrupt the stem elongation of A. philoxeroides in
aquatic habitats. In summary, this study shows that inhibiting
miR162 can effectively control the invasive capacity of A. philoxe-
roides in aquatic environments, and suggests that these findings
may have implications for the development of novel biocontrol
strategies against this invasive species.
5. Experimental Section
Plant Materials:The invasive population of A. philoxeroides used in
this study was collected in Zhuji, Zhejiang Province, China (120°20′E,
29°40′N). The collected materials were grown in nutrient-rich soil and
maintained in a greenhouse at Fudan University, Shanghai (121°29′E,
31°14′N). Stem fragments from a single individual plant were cut from
ramets with similar diameters and planted in plastic plates (dimensions:
upper diameter 16 cm, lower diameter 12.5 cm, height 13 cm), which
were filled with 1.5 L of a 1: 1 soil mixture (black soil: sand). Following
the appearance of the first two new leaves, plants of similar size were
individually transplanted into sand pots and grown under common
garden conditions in the greenhouse for a duration of 2 months prior to
the initiation of the two experimental treatments. For the submergence
treatment, the plants were fully immersed in 50 cm-deep water, while
the terrestrial control group received 1 L of water daily to maintain wet
but well-drained soil conditions. A. pungens were collected from Yunnan,
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China. The seeds were pre-germinated for 3 days before the seedlings
were individually transplanted into sand pots and maintained under the
same conditions as A. philoxeroides.ForArabidopsis thaliana plants, seeds
of the wild type Columbia-0 (Col-0), as well as various mutants includ-
ing hyl1-2 (SALK_064863),[31]ago1-27,[32 ]mir159abc (SAIL_430_F11 for
mir159a; SAIL_770_GO5 for mir159b; and SAIL_248_G11 for mir159c),[33]
mir164abc (Cs65828),[34]and myb33 myb65 (CS851168/myb33; crispr-
cas9/myb65)[7]were germinated on 1/2 Murashige and Skoog (MS)
medium and grown at 22 °C under a 16-h light/8-h dark photoperiod.
After 4 days on solid 1/2 MS medium, seedlings were transferred to
liquid MS medium, ensuring that the seedlings remained completely
submerged. They were allowed to grow for an additional ten days, after
which phenotypic observations were conducted.
Plasmid Construction:The vectors pTRV2 and pTRV2-GFP,[11]pro-
vided by Prof. Feng Li from Huazhong Agricultural University in China,
were utilized for the construction of the pTRV2-AphPDS, pTRV2-AphDCL1,
pTRV2-AphAGO1, pTRV2-AphMIR162a and pTRV2-AphMIR162b vectors.
DNA sequences were amplified from A. philoxeroides and subsequently
cloned into the pTRV2 vector. Details of the primers used for amplifica-
tion can be found in Table S2 (Supporting Information).
Inoculation of TRV-Based VIGS into A. philoxeroides:Overnight cultures
of Agrobacterium expressing the TRV2 vectors and the constitutive vector
of TRV1[12]were harvested by spinning, respectively, and the pellets were
re-suspended in a solution containing 10 mM MES (2-(N-morpholino)
ethanesulfonic acid), 10 mM MgCl2, and 200 mM acetosyringone (AS) to
a final optical density (OD600) of 1.5. Then, the TRV2 vector-containing
solution was mixed with the TRV1 plasmid-containing in a 1:1 ratio
with volumes. This agrobacterium suspension was then infiltrated into
each leaf of A. philoxeroides, with at least 10 plants inoculated for each
construct.
RNA Isolation, Library Preparation, and Sequencing:Stem samples
were collected from young (1-2 internodes) and old (3-4 internodes) plants
at multiple time points: 0, 3, 24, 48, 72, and 96 h, both in terrestrial con-
ditions and following waterlogging treatment. Each treatment and time
point consisted of three biological replicates, with each replicate includ-
ing three individual plants. Total RNA was extracted from the stems using
TRIzol reagent (Invitrogen, USA). The RNA concentration was determined
using a Qubit fluorometer (Thermo, USA), while the RNA quality was as-
sessed using a Qsep100 system with an R1 RNA Cartridge Kit (BiOptic
Inc., China). For small RNA sequencing, 1 μg of total RNA from each sam-
ple was used for library preparation, employing the VAHTS Small RNA
Library Prep Kit for Illumina V2 (Vazyme, NR811-01) according to the man-
ufacturer’s instructions. The purified small RNA libraries were sequenced
using the Illumina NovaSeq 6000 platform.
Analyses of Small RNA Sequencing Data:The sRNA-Seq libraries were
analyzed using computational methods reported in previous studies.[35,36]
Initially, the 3′adapters from all sequenced reads were clipped to obtain
the resulting small RNA reads. Reads shorter than 18 nucleotides were
discarded, and redundant reads were removed to generate a unique set
of reads with associated counts. To identify homologs of conserved miR-
NAs in Alternanthera philoxeroides, mature miRNA sequences from other
plant species were downloaded from miRBase (version 22),[37]and unique
mature miRNA sequences were obtained. These unique miRNAs were
subjected to alignment against the A. philoxeroides genome using NCBI
BLASTN (version 2.2.26),[38]with alignment parameters set to “-m 8 -e
0.01.” For every matched locus, flanking regions of 80, 130, and 180 nt both
downstream and upstream were extracted, and secondary structures for
these sequences were predicted using RNAfold.[39]Based on the predicted
fold-back structures and reported family members from other plants, the
existence of mature miRNAs located on the same arms of the hairpins
was examined. To validate these precursor sequences, MIRcheck was uti-
lized to ensure that the sequences met specific criteria: a maximum of
2 bulged nucleotides, fewer than or equal to five mismatches or asym-
metrically unpaired nucleotides, and no more than three continuous mis-
matches within the mature miRNA. Subsequently, small RNA sequencing
reads were aligned to the pre-miRNA candidates to observe the distribu-
tion of small RNA reads among these candidates, employing the criteria
outlined in a previous finding.[40]The frequencies of mature miRNAs in
the different small RNA sequencing profiles were calculated by aligning
the reads to the mature miRNAs using NCBI BLASTN (version 2.2.26)[37]
with parameters “-S 1 -m 8 -e 0.01,” and results were normalized to Reads
Per Ten Million (RPTM) sequencing tags. To compare expression levels of
miRNAs among the different sample groups, the edgeR software package
was utilized.[41]Specifically, comparisons were made using young and old
stem samples from both terrestrial and waterlogged conditions at time
points of 3, 24, 48, 72, and 96 h against the control samples collected at 0
h. miRNA was considered significantly deregulated if the corrected p-value
was less than 0.05.
miRNA Detection By Stem-Loop RT-qPCR:A quantity of 0.5 μgof
DNase I-treated RNA was subjected to first-strand cDNA synthesis in 20 μL
reaction with RT buffer (50 mmol L−1 Tris-Cl pH 8.3, 75 mmol L−1KCl,
3 mmol L−1 MgCl2,2UμL−1 RNase inhibitor), 1 μL dNTPs (10 mM),
SuperScript IV reverse transcriptase and 1μM each stem-loop primers de-
signed for the miRNAs of interest and the U6. The reaction mixture was
incubated at 16 °C for 30 min (this step allows the stem-loop primer to
anneal), followed by pulsed RT of 60 cycles at 30 °C for 30 s, 42 °Cfor
30 s, and 50 °C for 1 s, the reverse transcriptase was inactivated by heat-
ing at 85 °C for 5 min as described previously.[42,43]The resulting cDNA
was diluted with RNase-free water to the concentration suitable for qPCR,
typically 1:5 to 1:10.
Quantitative Real-Time PCR:Total RNA was extracted using TRIzol.
The PrimeScript II Reverse Transcriptase (Takara, 2690B) was used to
reverse-transcribe RNA into cDNA following the manufacturer’s proto-
col, then the cDNAs were subjected to real-time qPCR using the CFX
Connect Real-Time System (Bio-Rad, USA). Each reaction was replicated
three times. The relative expression levels were calculated using the 2−ΔΔCt
method. The primers are listed in Table S2 (Supporting Information).
Preparation of Tetrahedral DNA Framework (TDF) Nanoparticle:The
tetrahedral DNA framework (TDF) with an edge length of 13 bp was syn-
thesized by mixing four designed strands ((T13-a, b, c, d, see detailed
sequences in Table S2, Supporting Information.) synthesized by Sangon
Biotech in equimolar ratios in TM buffer (10 mM Tris-HCl, 5 mM MgCl2,
pH 8.0), following the previously reported methods.[23,24]The solution
was heated to 95 °C for 10 min and then quickly cooled to 4 °Cfor30min.
For TDF aimed at loading 1, 2, 3, or 4 sticky ends for miR162-as, T13-a, b,
c, and d were replaced with 10T-T13-a, b, c, and d.
Assembly of miR162-as onto TDFs:First, the experimental steps out-
lined above to synthesize TDFs with suspended polyT sticky ends were
followed. PolyA-miR162-as was then added to the TDFs in a calculated
molar ratio and annealed using the same procedure. Subsequently, PolyA-
miR162-as with polyA tails were site-specifically coupled to TDFs through
hybridization with free polyT sequences displayed at specific vertex sites.
This approach allowed to generation of TDFs containing one, two, three,
or four miR162-as.
Infiltration of Leaves with TDF-Loaded miR162-as:In brief, the process
involves the following steps: using a sterile needle to make a short incision
on the abaxial surface of the A. philoxeroides leaf lamina. It is crucial that
the incision is shorter than the tip of a syringe to minimize damage to
the plant tissue. Then, using a 1-mL needleless syringe, gently infiltrate
100 μL of the prepared nanostructure solutions into the incision site. After
the injection, immediately submerge the treated plants in water for ten
days. Following this period, compare the elongation of the stems between
treated and control plants. Measure the stem lengths to assess the impact
of the nanostructure treatments on plant growth.
Native Polyacrylamide Gel Electrophoresis:The successful assembly of
TDF and TDF loaded with miR162-as were characterized using 10% native
polyacrylamide gel electrophoresis (PAGE) with 1×TAE buffer running at
85 V for ≈80 min in the electrophoresis apparatus instrument. Then, the
gels were stained with 1 ×GelRed nucleic acid dye for ≈2 min and scanned
under UV light. Image analysis was performed with the software Image J.
Atomic Force Microscopy:For AFM images of TDFs, 10 μLof25nM
TDFs were dropped onto the mica surface for 10 min. 20 μL1×TM buffer
was dropped onto the mica surface. The image of TDFs was obtained by
Bruker multimode 8.
Dynamic Light Scattering Analysis:Three hundred microliter TDF so-
lution (1 μM) was added into a plastic cuvette, then the hydrodynamic
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diameters were measured by a Malvern Zetasizer Nano-ZS instrument
(Malvern Instruments, UK) equipped with a He–Ne laser (633 nm).
Microscopy:GFP fluorescence microscopy analyses were carried out
with an Olympus FV3000 laser confocal microscope, excitation and emis-
sion wavelengths were 488 nm and 500 to 550 nm respectively. Cy3 flu-
orescence signals (emission range from 570 to 620 nm) were collected
by Lecia stellaris 5 under the 561 nm excitation wavelength. The captured
images were processed using Adobe Photoshop and Image J software.
Scanning Electron Microscope (SEM):Stems collected from relative A.
philoxeroides were carefully placed on conductive tape for analysis using
a Hitachi TM3000 scanning electron microscope. The parameters were
set as follows: Accelerating Voltage =15 000 Volt, Magnification =200,
Brightness =2243, Contrast =4095. The captured pictures were used to
measure the lengths of stem epidermal cells through Image J software.
Predicting the Cis-Regulatory Elements Located in Promoter Regions:
The PlantCARE (https://www.bioinformatics.psb.ugent.be/webtools/
plantcare/html/) online database was used to predict cis-regulatory
elements located in the promoter sequences of MIR162 genes in A.
philoxeroides and A. pungens.
Quantification and Statistical Analysis:Statistical methods for assess-
ing peak overlap included one-sided permutation overlap tests or two-
sided Fisher’s exact tests, respectively. All boxplots, bar plots, and line
charts were generated by R or GraphPad Prism 9.5. Statistical significance
was determined by one-way ANOVA or Student’s t-test.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors appreciate Prof. Feng Li for providing the pTRV plasmids.
This work was supported by the National Key R&D Program of China
(2021YFC2600102 to J.Y.), the National Natural Science Foundation of
China (32025005 to B.Z., 22377076 to H.Z.), the China Postdoctoral Sci-
ence Foundation (2023M732270 to E.K.).
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
Q.H. and E.Q. contributed equally to this work and co-first authors. B.Z.
and J.Y. conceptualized the study. Q.H. performed most experiments. E.Q.
and H. Z. finished the preparation of nanoparticles. X. L. and Y.Z. fin-
ished miRNome analysis. R.Q. and Q.T. helped with sample preparation
for small RNA seq analysis. B.Z. wrote the manuscript, and Q.H., E.Q.,
Y.Z., H. Z, and J.Y. revised the manuscript.
Data Availability Statement
The data that support the findings of this study are openly available
in NCBI GEO at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=
GSE283087, reference number 283087.
Keywords
Alternanthera philoxeroides , invasive plants, miRNA, phenotypic plasticity
Received: December 12, 2024
Revised: February 15, 2025
Published online: March 31, 2025
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