Bacteriophage T4 as a Protein-Based, Adjuvant- and Needle-Free, Mucosal Pandemic Vaccine Design Platform.

The COVID-19 pandemic has transformed vaccinology. Rapid deployment of mRNA vaccines has saved countless lives. However, these platforms have inherent limitations including lack of durability of immune responses and mucosal immunity, high cost, and thermal instability. These and uncertainties about the nature of future pandemics underscore the need for exploring next-generation vaccine platforms. Here, we present a novel protein-based, bacteriophage T4 platform for rapid design of efficacious vaccines against bacterial and viral pathogens. Full-length antigens can be displayed at high density on a 120 × 86 nm phage capsid through nonessential capsid binding proteins Soc and Hoc. Such nanoparticles, without any adjuvant, induce robust humoral, cellular, and mucosal responses when administered intranasally and confer sterilizing immunity. Combined with structural stability and ease of manufacture, T4 phage provides an excellent needle-free, mucosal pandemic vaccine platform and allows equitable vaccine access to low- and middle-income communities across the globe.

T4 bacteriophage nanoparticles engineered through CRISPR provide a versatile platform for rapid development of flu mucosal vaccines.

Vaccines that trigger mucosal immune responses at the entry portals of pathogens are highly desired. Here, we showed that antigen-decorated nanoparticle generated through CRISPR engineering of T4 bacteriophage can serve as a universal platform for the rapid development of mucosal vaccines. Insertion of Flu viral M2e into phage T4 genome through fusion to Soc (Small Outer Capsid protein) generated a recombinant phage, and the Soc-M2e proteins self-assembled onto phage capsids to form 3M2e-T4 nanoparticles during propagation of T4 in E. coli. Intranasal administration of 3M2e-T4 nanoparticles maintains antigen persistence in the lungs, resulting in increased uptake and presentation by antigen-presenting cells. M2e-specific secretory IgA, effector (TEM), central (TCM), and tissue-resident memory CD4+ T cells (TRM) were efficiently induced in the local mucosal sites, which mediated protections against divergent influenza viruses. Our studies demonstrated the mechanisms of immune protection following 3M2e-T4 nanoparticles vaccination and provide a versatile T4 platform that can be customized to rapidly develop mucosal vaccines against future emerging epidemics.

Design of bacteriophage T4-based artificial viral vectors for human genome remodeling.

Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an additional category of nanomaterial that could potentially transform gene therapies and personalized medicine.

Understanding Bacteriophage Tail Fiber Interaction with Host Surface Receptor: The Key "Blueprint" for Reprogramming Phage Host Range.

Bacteriophages (phages), as natural antibacterial agents, are being rediscovered because of the growing threat of multi- and pan-drug-resistant bacterial pathogens globally. However, with an estimated 1031 phages on the planet, finding the right phage to recognize a specific bacterial host is like looking for a needle in a trillion haystacks. The host range of a phage is primarily determined by phage tail fibers (or spikes), which initially mediate reversible and specific recognition and adsorption by susceptible bacteria. Recent significant advances at single-molecule and atomic levels have begun to unravel the structural organization of tail fibers and underlying mechanisms of phage-host interactions. Here, we discuss the molecular mechanisms and models of the tail fibers of the well-characterized T4 phage's interaction with host surface receptors. Structure-function knowledge of tail fibers will pave the way for reprogramming phage host range and will bring future benefits through more-effective phage therapy in medicine. Furthermore, the design strategies of tail fiber engineering are briefly summarized, including machine-learning-assisted engineering inspired by the increasingly enormous amount of phage genetic information.

Bacteriophage T4 as a nanovehicle for delivery of genes and therapeutics into human cells.

The ability to deliver therapeutic genes and biomolecules into a human cell and restore a defective function has been the holy grail of medicine. Adeno-associated viruses and lentiviruses have been extensively used as delivery vehicles, but their capacity is limited to one (or two) gene(s). Bacteriophages are emerging as novel vehicles for gene therapy. The large 120 × 86-nm T4 capsid allows engineering of both its surface and its interior to incorporate combinations of DNAs, RNAs, proteins, and their complexes. In vitro assembly using purified components allows customization for various applications and for individualized therapies. Its large capacity, cell-targeting capability, safety, and inexpensive manufacturing could open unprecedented new possibilities for gene, cancer, and stem cell therapies. However, efficient entry into primary human cells and intracellular trafficking are significant barriers that must be overcome by gene engineering and evolution in order to translate phage-delivery technology from bench to bedside.

A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

CRISPR Engineering of Bacteriophage T4 to Design Vaccines Against SARS-CoV-2 and Emerging Pathogens.

The COVID-19 pandemic brought to the fore the urgent need for vaccine design and delivery platforms that can be rapidly deployed for manufacture and distribution. Though the mRNA and adenoviral vector platforms have been enormously successful to control SARS-CoV-2 viral infections, it is unclear if this could be replicated against more complex pathogens or the emerging variants. Recently, we described a "universal" platform that can incorporate multiple vaccine targets into the same nanoparticle scaffold by CRISPR engineering of bacteriophage T4. A T4-COVID vaccine designed with this technology elicited broad immunogenicity and complete protection against virus challenge in a mouse model. Here, we describe the detailed methodology to generate recombinant bacteriophage T4 backbones using CRISPR that can also be broadly applicable to other bacteriophages that abundantly pervade the Earth.

Engineering T4 Bacteriophage for In Vivo Display by Type V CRISPR-Cas Genome Editing.

Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether, in vivo, foreign peptides and proteins to T4 capsid. Our data show that while these could be successfully expressed and displayed during the phage infection, shorter peptides are present at a much higher copy number than full-length proteins. However, the copy number of the latter could be elevated by driving the expression of the transgene using the strong T7 RNA polymerase expression system. This CRISPR-inspired approach has the potential to expand the application of phages to various basic and translational research projects.

A universal bacteriophage T4 nanoparticle platform to design multiplex SARS-CoV-2 vaccine candidates by CRISPR engineering.

A "universal" platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (TH1) and TH2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.

Bacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair.

Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications.

A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering.

A "universal" vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both T H 1 and T H 2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

Covalent Modifications of the Bacteriophage Genome Confer a Degree of Resistance to Bacterial CRISPR Systems.

The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both.IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.

Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells.

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus "piggy-backed" on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a "driver" to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.

A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells.

Development of safe and efficient nanoscale vehicles that can deliver large molecular cargos into human cells could transform future human therapies and personalized medicine. Here, we design a hybrid viral vector composed of a prokaryotic virus (bacteriophage T4) and a eukaryotic virus [adeno-associated virus (AAV)]. The small 25-nm AAV is attached to the large 120 nm × 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc and Hoc. AAV "piggy-backed" on T4 capsid, by virtue of its natural ability to enter human cells acted as an efficient "driver," delivering the largest payloads of foreign DNA (up to 170 kb) and protein (up to 1025 molecules) reported to date, and elicited robust immune responses in mice against flu and plague pathogens and conferred complete protection against lethal pneumonic plague challenge. The T4-AAV represents a unique platform for assembly of natural building blocks into potential therapeutics against genetic and infectious diseases.

Bacteriophage T4 nanoparticles for vaccine delivery against infectious diseases.

Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.

A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague.

Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

A Bivalent Anthrax-Plague Vaccine That Can Protect against Two Tier-1 Bioterror Pathogens, Bacillus anthracis and Yersinia pestis.

Bioterrorism remains as one of the biggest challenges to global security and public health. Since the deadly anthrax attacks of 2001 in the United States, Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, gained notoriety and were listed by the CDC as Tier-1 biothreat agents. Currently, there is no Food and Drug Administration-approved vaccine against either of these threats for mass vaccination to protect general public, let alone a bivalent vaccine. Here, we report the development of a single recombinant vaccine, a triple antigen consisting of all three target antigens, F1 and V from Y. pestis and PA from B. anthracis, in a structurally stable context. Properly folded and soluble, the triple antigen retained the functional and immunogenicity properties of all three antigens. Remarkably, two doses of this immunogen adjuvanted with Alhydrogel® elicited robust antibody responses in mice, rats, and rabbits and conferred complete protection against inhalational anthrax and pneumonic plague. No significant antigenic interference was observed. Furthermore, we report, for the first time, complete protection of animals against simultaneous challenge with Y. pestis and the lethal toxin of B. anthracis, demonstrating that a single biodefense vaccine can protect against a bioterror attack with weaponized B. anthracis and/or Y. pestis. This bivalent anthrax-plague vaccine is, therefore, a strong candidate for stockpiling, after demonstration of its safety and immunogenicity in human clinical trials, as part of national preparedness against two of the deadliest bioterror threats.

Higher-Ordered Actin Structures Remodeled by Arabidopsis ACTIN-DEPOLYMERIZING FACTOR5 Are Important for Pollen Germination and Pollen Tube Growth.

Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN-DEPOLYMERIZING FACTORs (ADFs) typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs (ADF5 and ADF9) evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neofunctionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B (LatB) treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochemical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.