Oryza sativa actin‐interacting protein 1 is required for rice growth by promoting actin turnover

Rapid actin turnover is essential for numerous actin‐based processes. However, how it is precisely regulated remains poorly understood. Actin‐interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin‐depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate‐limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down‐regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over‐expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF‐mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real‐time visualization of single filament dynamics showed that OsAIP1 enhanced ADF‐mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off‐rate were enhanced in OsAIP1 over‐expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F‐actin levels in plants.     

Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α‐helix 3 and forming an actin binding site together with the N‐terminus are essential for both G‐ and F‐actin binding. The basic residues on the β‐strand 5 (K82/A) and the α‐helix 4 (R135/A, R137/A) form another actin binding site that is important for F‐actin binding. Using transient expression of CFP‐tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G‐actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Regulation of a wheat actin-depolymerizing factor during cold acclimation

We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.     

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.     

TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust

Calcineurin B‐like interacting protein kinase (CIPKs) has been shown to be required for biotic stress tolerance of plants in plant‐pathogen interactions. However, the roles of CIPKs in immune signalling of cereal crops and an in‐depth knowledge of substrates of CIPKs in response to biotic stress are under debate. In this study, we identified and cloned a CIPK homologue gene TaCIPK10 from wheat. TaCIPK10 was rapidly induced by Puccinia striiformis f. sp. tritici (Pst) inoculation and salicylic acid (SA) treatment. In vitro phosphorylation assay demonstrated that the kinase activity of TaCIPK10 is regulated by Ca²⁺ and TaCBL4. Knockdown TaCIPK10 significantly reduced wheat resistance to Pst, whereas TaCIPK10 overexpression resulted in enhanced wheat resistance to Pst by the induction of defense response in different aspects, including hypersensitive cell death, ROS accumulation and pathogenesis‐relative genes expression. Moreover, TaCIPK10 physically interacted with and phosphorylated TaNH2, which was homologous to AtNPR3/4. Silencing of TaNH2 in wheat resulted in enhanced susceptibility to the avirulent Pst race, CYR23, indicating its positive role in wheat resistance. Our results demonstrate that TaCIPK10 positively regulate wheat resistance to Pst as molecular links between of Ca²⁺ and downstream components of defense response and TaCIPK10 interacts with and phosphorylates TaNH2 to regulate wheat resistance to Pst.     

Genetic and Molecular Mapping of Stripe Rust Resistance Genes in Wheat Cultivar Zhongliang 12

Stripe rust, caused by Puccinia striiformis f.sp. tritici (Pst), is one of the most widespread and destructive diseases of wheat worldwide. Resistance breeding is constantly pursued for decades to tackle the variations of prevalent Pst races. Zhongliang 12 has strong resistance to abiotic stresses, wide adaptability, higher resistance to stripe rust and excellent biological characteristics. To identify the resistance gene(s) against stripe rust, Zhongliang 12 was crossed with stripe rust susceptible genotype Mingxian 169, and F₁, F₂, F_2 : 3 and BC₁ progenies were tested with Chinese Pst race CYR30 and CYR31 in seedling stage in greenhouse. Zhongliang 12 possessed different dominant genes for resistance to each race. Linkage maps were constructed with four simple sequence repeats (SSRs) markers, Xwmc695, Xcfd20, Xbarc121 and Xbarc49, for the gene on wheat chromosome 7AL conferring resistance to CYR30 (temporarily designated as Yrzhong12‐1) with genetic distance ranging from 3.1 to 10.8 cM and four SSR markers, Xpsp3003, Xcfd2129, Xwmc673 and Xwmc51, for the gene on wheat chromosome 1AL conferring resistance to CYR31 (temporarily designated as Yrzhong12‐2) with genetic distance ranging from 3.9 cM to 9.3 cM. The molecular markers closely linked to each gene should be useful in marker‐assisted selection in breeding programmes for against stripe rust.     

Molecular Detection of Stripe Rust Resistance Gene(s) in 115 Wheat Cultivars (lines) from the Yellow and Huai River Valley Wheat Region

Stripe rust (yellow rust), caused by Puccinia striiformis f.sp. tritici (Pst), is a serious disease of wheat worldwide, including China. Growing resistant cultivars is the most cost‐effective and environmentally friendly approach to control the disease. To assess the stripe rust resistance in commercial wheat cultivars and advanced lines in the Yellow and Huai River Valley Wheat Region, 115 wheat cultivars (lines) collected from 13 provinces in this region were evaluated with the most prevalent Chinese Pst races CYR32, CYR33 and the new race V26 at seedling stage. In addition, these wheat entries were inoculated with the mixed races of CYR32 and CYR33 at the adult‐plant stage in the field. The results indicated that 53 (46.1%) cultivars (lines) had all‐stage resistance to all the three races, and 16 (13.9%) cultivars (lines) showed adult‐plant resistance. The possible stripe rust resistance genes in these entries were postulated by the closely linked markers of all‐stage resistance genes Yr5, Yr9, Yr10, Yr15 and Yr26 and adult‐plant resistance gene Yr18. Molecular analysis indicated that resistance genes Yr5, Yr9, Yr10, Yr18 and Yr26 were found in 5 (4.3%), 38 (33.0%), 1 (0.9%), 2 (1.7%) and 8 (7.0%) entries, respectively. No entry was found to carry the Yr15 gene. In future breeding programs, Yr5, Yr15 and Yr18 should be used to pyramid with other effective genes to develop wheat cultivars with high‐level and durable resistance to stripe rust, whereas Yr9, Yr10 and Yr26 should not be used or used in a limited way due to the virulent races present in China.     

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth

Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.     

How tropomyosin regulates lamellipodial actin‐based motility: a combined biochemical and reconstituted motility approach

At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott–Aldrich syndrome protein (N‐WASP)‐functionalised beads, which is based on the sole formation of a dendritic array of actin‐related protein (Arp)2/3‐branched filaments. Actin‐depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N‐WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.     

Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici

MicroRNAs (miRNAs) play very important roles in plant defense responses. However, little is known about their roles in the susceptibility interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). In this study, two miRNA libraries were constructed from the leaves of the cultivar Xingzi 9104 inoculated with the virulent Pst race CYR32 and sterile water, respectively. A total of 1316 miRNA candidates, including 173 known miRNAs that were generated from 98 pre‐miRNAs, were obtained. The remaining 1143 miRNA candidates included 145 conserved and 998 wheat‐specific miRNAs that were generated from 87 and 1088 pre‐miRNAs, respectively. The 173 known and 145 conserved miRNAs were sub‐classified into 63 miRNA families. The target genes of wheat miRNAs were also confirmed using degradome sequencing technology. Most of the annotated target genes were related to signal transduction or energy metabolism. Additionally, we found that miRNAs and their target genes form complicated regulation networks. The expression profiles of miRNAs and their corresponding target genes were further analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the results indicate that some miRNAs are involved in the compatible wheat‐Pst susceptibility interaction. Importantly, tae‐miR1432 was highly expressed when wheat was challenged with CYR32, and the corresponding target gene, predicted to be a calcium ion‐binding protein, also exhibited upregulated expression but a divergent expression trend. PC‐3P‐7484, a specific wheat miRNA, was highly expressed in the wheat response to Pst infection, while the expression of the corresponding target gene ubiquillin was dramatically downregulated. These data provide the foundation for evaluating the important regulatory roles of miRNAs in wheat‐Pst susceptibility interaction.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

Genetic Analysis and Molecular Mapping of a Stripe Rust Resistance Gene in Chinese Wheat Differential Guinong 22

Stripe rust, caused by Puccinia striiformis f.sp. tritici (Pst), is one of the most damaging diseases of wheat worldwide, especially in China. Growing resistant cultivars is the most effective approach to control the disease, but few effective resistance genes are available. Guinong 22, one of the wheat cultivars used for differentiated Chinese race of the pathogen, has unknown resistance gene(s) to stripe rust. Genetic analysis, molecular mapping and allelic analysis were used in this study to determine the inheritance and chromosomal location of the gene(s) in Guinong 22 with the most prevalent Pst race CYR33. Genetic analysis indicated that a single recessive gene yrGn22 confers the resistance to CYR33. A total of 450 simple sequence repeat (SSR) primer pairs and 31 pairs of sequence‐tagged site (STS) or conserved primers were selected to screen the resistant bulk and susceptible bulk as well as the parents. Seven polymorphic SSR markers and two STS markers were then used to genotype 113 F₂ individual plants. Linkage analysis indicated that all nine markers were linked to yrGn22, with genetic distances ranging from 2.2 to 11.1 cM. Based on the chromosomal locations of the linked markers, yrGn22 was located on wheat chromosome 1B near the centromere. The pedigree, common markers, chromosome location, resistance and allelism tests indicated that yrGn22 is either linked to Yr26 or possibly the same gene.     

Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments

Actin depolymerizing factor (ADF)/cofilin enhances turnover of actin filaments by severing and depolymerizing filaments. A number of proteins functionally interact with ADF/cofilin to modulate the dynamics of actin filaments. Actin-interacting protein 1 (AIP1) has emerged as a conserved WD-repeat protein that specifically enhances ADF/cofilin-induced actin dynamics. Interaction of AIP1 with actin was originally characterized by a yeast two-hybrid system. However, biochemical studies revealed its unique activity on ADF/cofilin-bound actin filaments. AIP1 alone has negligible effects on actin filament dynamics, whereas in the presence of ADF/cofilin, AIP1 enhances filament fragmentation by capping ends of severed filaments. Studies in model organisms demonstrated that AIP1 genetically interacts with ADF/cofilin and participates in several actin-dependent cellular events. The crystal structure of AIP1 revealed its unique structure with two seven-bladed beta-propeller domains. Thus, AIP1 is a new class of actin regulatory proteins that selectively enhances ADF/cofilin-dependent actin filament dynamics.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite

Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.     

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response

The development of improved plant nucleotide‐binding, leucine‐rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3^A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3^A2/F2. PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race‐specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.     

A polysaccharide deacetylase from Puccinia striiformis f. sp. tritici is an important pathogenicity gene that suppresses plant immunity

The cell wall of filamentous fungi, comprised of chitin, polysaccharide and glycoproteins, maintains the integrity of hyphae and protect them from defence responses by potential host plants. Here, we report that one polysaccharide deacetylase of Puccinia striiformis f. sp. tritici (Pst), Pst_13661, suppresses Bax‐induced cell death in plants and Pst_13661 is highly induced during early stages of the interaction between wheat and Pst. Importantly, the transgenic wheat expressing the RNA interference (RNAi) construct of Pst_13661 exhibits high resistance to major Pst epidemic races CYR31, CYR32 and CYR33 by inhibiting growth and development of Pst, indicating that Pst_13661 is an available pathogenicity factor and is a potential target for generating broad‐spectrum resistance breeding material of wheat. It forms a homo‐polymer and has high affinity for chitin and germ tubes of Pst compared with the control. Besides, Pst_13661 suppresses chitin‐induced plant defence in plants. Hence, we infer that Pst_13661 may modify the fungal cell wall to prevent recognition by apoplastic surveillance systems in plants. This study opens new approaches for developing durable disease‐resistant germplasm by disturbing the growth and development of fungi and develops novel strategies to control crop diseases.