Secretome Analysis Identifies Potential Pathogenicity/Virulence Factors of Tilletia indica,a Quarantined Fungal Pathogen Inciting Karnal Bunt Disease in Wheat

Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity / virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI‐TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host‐derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity / virulence factors homologs that were further subjected to sequence‐ and structure‐based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity / virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies.     

Characterization of Extended-Host-Range Pseudo-T-Even Bacteriophage Kpp95 Isolated on Klebsiella pneumoniae

Kpp95, isolated on Klebsiella pneumoniae, is a bacteriophage with the morphology of T4-type phages and is capable of rapid lysis of host cells. Its double-stranded genomic DNA (ca. 175 kb, estimated by pulsed-field gel electrophoresis) can be cut only by restriction endonucleases with a cleavage site flanked either by A and T or by T, as tested, suggesting that it contains the modified derivative(s) of G and/or C. Over 26 protein bands were visualized upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virion proteins. N-terminal sequencing indicated that the most abundant band (46 kDa) is the major coat protein (gp23) which has been cleaved from a signal peptide likely with a length similar to that of T4. Phylogenetic analyses based on the sequences of the central region (263 amino acid residues) of gp23 and the full length of gp18 and gp19 placed Kpp95 among the pseudo-T-even subgroup, most closely related to the coliphage JS98. In addition to being able to lyse many extended-spectrum β-lactamase strains of K. pneumoniae, Kpp95 can lyse Klebsiella oxytoca, Enterobacter agglomerans, and Serratia marcescens cells. Thus, Kpp95 deserves further studies for development as a component of a therapeutic cocktail, owing to its high efficiencies of host lysis plus extended host range.     

Development of Seed Immunoblot Binding Assay for Detection of Karnal bunt (Tilletia indica) of Wheat

Polyclonal antisera were produced in albino white rabbits against intact teliospores of Karnal bunt (Tilletia indica). The Immunoprobe generated was used for the development of Immunoblot binding assay for detecting Karnal bunt (KB) infections in wheat seed samples. The antiserum reacted strongly with intact teliospores of T. indica, Pantnagar isolate in agglutination reaction. Wheat grains with different grades of infection could be readily detected by Seed Immunoblot Binding Assay (SIBA). Karnal bunt infected wheat seeds when kept for vigour testing on nitrocellulose paper, formed a coloured imprint after the paper was immunoprocessed. The SIBA would not only be a better Indication of teliospores load on seed but also quality of seed in terms of vigour. This method is expected to be useful in routine monitoring of wheat lots for the presence of KB teliospores.     

Two stripe rust effectors impair wheat resistance by suppressing import of host Fe–S protein into chloroplasts

Several effectors from phytopathogens usually target various cell organelles to interfere with plant defenses, and they generally contain sequences that direct their translocation into organelles, such as chloroplasts. In this study, we characterized a different mechanism for effectors to attack chloroplasts in wheat (Triticum aestivum). Two effectors from Puccinia striiformis f. sp. tritici (Pst), Pst_4, and Pst_5, inhibit Bax-mediated cell death and plant immune responses, such as callose deposition and reactive oxygen species (ROS) accumulation. Gene silencing of the two effectors induced significant resistance to Pst, demonstrating that both effectors function as virulence factors of Pst. Although these two effectors have low sequence similarities and lack chloroplast transit peptides, they both interact with TaISP (wheat cytochrome b6–f complex iron–sulfur subunit, a chloroplast protein encoded by nuclear gene) in the cytoplasm. Silencing of TaISP impaired wheat resistance to avirulent Pst and resulted in less accumulation of ROS. Heterogeneous expression of TaISP enhanced chloroplast-derived ROS accumulation in Nicotiana benthamiana. Co-localization in N. benthamiana and western blot assay of TaISP content in wheat chloroplasts show that both effectors suppressed TaISP from entering chloroplasts. We conclude that these biotrophic fungal effectors suppress plant defenses by disrupting the sorting of chloroplast protein, thereby limiting host ROS accumulation and promoting fungal pathogenicity.

Despite the lack of chloroplast transit peptide, rust effectors affect chloroplast-mediated defenses by suppressing import of host Fe–S protein to chloroplast to promote pathogenicity of stripe rust.     

Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica

The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4''s genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4''s pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant''s jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4- and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium.     

Comparative pathogenicity of Fusarium graminearum isolates from China revealed by wheat coleoptile and floret inoculations

Fusarium head blight (FHB) or scab caused by Fusarium species is an economically important disease on small grain cereal crops worldwide. Accurate assessments of the pathogenicity of fungal isolates is a key obstacle toward a better understanding of the Fusarium-wheat scab system. In this study, a new laboratory method for inoculation of wheat coleoptiles was developed, which consists of cutting off the coleoptile apex, covering the cut apex with a piece of filter paper soaked in conidial suspension, and measuring the lengths of brown lesions 7 days post inoculation. After coleoptile inoculation, distinct brown lesions in the diseased stems were observed, in which the presence of the fungus was verified by PCR amplification with F.␣graminearum Schwable-specific primers. Coleoptile inoculation of six wheat varieties indicated that a highly susceptible wheat variety was more suitable as a differentiating host for the pathogenicity assay. Analysis of the coleoptiles inoculated with a set of 58 different isolates of F. graminearum showed a significant difference in the lengths of the lesions, forming the basis by which pathogenicity of the isolates was assessed. Field inoculation of florets of three wheat varieties over 2 years revealed significant differences in pathogenicity among the 58 isolates, and that the highly resistant and highly susceptible wheat varieties were more appropriate and stable for pathogenicity assessment in field trials. Comparative analyses of eight inoculation experiments of wheat with 58 F. graminearum isolates showed significant direct linear correlations (P<0.001) between coleoptile and floret inoculations. These results indicate that the wheat coleoptile inoculation is a simple, rapid and reliable method for pathogenicity studies of F.␣graminearum in wheat.     

Necrotrophic Effector Epistasis in the Pyrenophora tritici-repentis-Wheat Interaction

Pyrenophora tritici-repentis, the causal agent of tan spot disease of wheat, mediates disease by the production of host-selective toxins (HST). The known toxins are recognized in an ‘inverse’ gene-for-gene manner, where each is perceived by the product of a unique locus in the host and recognition leads to disease susceptibility. Given the importance of HSTs in disease development, we would predict that the loss of any of these major pathogenicity factors would result in reduced virulence and disease development. However, after either deletion of the gene encoding the HST ToxA or, reciprocally, heterologous expression of ToxA in a race that does not normally produce the toxin followed by inoculation of ToxA-sensitive and insensitive wheat cultivars, we demonstrate that ToxA symptom development can be epistatic to other HST-induced symptoms. ToxA epistasis on certain ToxA-sensitive wheat cultivars leads to genotype-specific increases in total leaf area affected by disease. These data indicate a complex interplay between host responses to HSTs in some genotypes and underscore the challenge of identifying additional HSTs whose activity may be masked by other toxins. Also, through mycelial staining, we acquire preliminary evidence that ToxA may provide additional benefits to fungal growth in planta in the absence of its cognate recognition partner in the host.     

Genes <Emphasis Type="Italic">WHEAT FRIZZY PANICLE</Emphasis> and <Emphasis Type="Italic">SHAM RAMIFICATION 2</Emphasis> independently regulate differentiation of floral meristems in wheat

Background

Inflorescences of wheat species, spikes, are characteristically unbranched and bear one sessile spikelet at a spike rachis node. Development of supernumerary spikelets (SSs) at rachis nodes or on the extended rachillas is abnormal. Various wheat morphotypes with altered spike morphology, associated with the development of SSs, present an important genetic resource for studies on genetic regulation of wheat inflorescence development.

Results

Here we characterized diploid and tetraploid wheat lines of various non-standard spike morphotypes, which allowed for identification of a new mutant allele of the WHEAT FRIZZY PANICLE (WFZP) gene that determines spike branching in diploid wheat Ttiticum monococcum L. Moreover, we found that the development of SSs and spike branching in wheat T. durum Desf. was a result of a wfzp-A/TtBH-A1 mutation that originated from spontaneous hybridization with T. turgidum convar. сompositum (L.f.) Filat. Detailed characterization of the false-true ramification phenotype controlled by the recessive sham ramification 2 (shr2) gene in tetraploid wheat T. turgidum L. allowed us to suggest putative functions of the SHR2 gene that may be involved in the regulation of spikelet meristem fate and in specification of floret meristems. The results of a gene interaction test suggested that genes WFZP and SHR2 function independently in different processes during spikelet development, whereas another spike ramification gene(s) interact(s) with SHR2 and share(s) common functions.

Conclusions

SS mutants represent an important genetic tool for research on the development of the wheat spikelet and for identification of genes that control meristem activities. Further studies on different non-standard SS morphotypes and wheat lines with altered spike morphology will allow researchers to identify new genes that control meristem identity and determinacy, to elucidate the interaction between the genes, and to understand how these genes, acting in concert, regulate the development of the wheat spike.

     

In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.     

Heterochronic development of the floret meristem determines grain number per spikelet in diploid, tetraploid and hexaploid wheats

Background and Aims

The inflorescence of grass species such as wheat, rice and maize consists of a unique reproductive structure called the spikelet, which is comprised of one, a few, or several florets (individual flowers). When reproductive growth is initiated, the inflorescence meristem differentiates a spikelet meristem as a lateral branch; the spikelet meristem then produces a floret meristem as a lateral branch. Interestingly, in wheat, the number of fertile florets per spikelet is associated with ploidy level: one or two florets in diploid, two or three in tetraploid, and more than three in hexaploid wheats. The objective of this study was to identify the mechanisms that regulate the architecture of the inflorescence in wheat and its relationship to ploidy level.

Methods

The floral anatomy of diploid (Triticum monococcum), tetraploid (T. turgidum ssp. durum) and hexaploid (T. aestivum) wheat species were investigated by light and scanning electron microscopy to describe floret development and to clarify the timing of the initiation of the floret primordia. In situ hybridization analysis using Wknox1, a wheat knotted1 orthologue, was performed to determine the patterning of meristem formation in the inflorescence.

Key Results

The recessive natural mutation of tetraploid (T. turgidum ssp. turgidum) wheat, branching head (bh), which produces branched inflorescences, was used to demonstrate the utility of Wknox1 as a molecular marker for meristematic tissue. Then an analysis of Wknox1 expression was performed in diploid, tetraploid and hexaploid wheats and heterochronic development of the floret meristems was found among these wheat species.

Conclusions

It is shown that the difference in the number of floret primordia in diploid, tetraploid and hexaploid wheats is caused by the heterochronic initiation of floret meristem development from the spikelet meristem.Key words: Triticum, wheat, inflorescence, spikelet, floret, meristem, heterochrony, heterochronic development, knotted1, polyploidy     

Asynchronous flowering and within-plant flowering diversity in wheat and the implications for crop resilience to heat

Background and Aims

Self-pollination dominates in wheat, with a small level of out-crossing due to flowering asynchrony and male sterility. However, the timing and synchrony of male and female flowering in wheat is a crucial determinant of seed set and may be an important factor affecting gene flow and resilience to climate change. Here, a methodology is presented for assessing the timing and synchrony of flowering in wheat, Triticum aestivum.

Methods

From the onset of flowering until the end of anthesis, the anther and stigma activity of each floret was assessed on the first five developing ears in potted plants grown under ambient conditions and originating from ‘Paragon’ or ‘Spark-Rialto’ backgrounds. At harvest maturity, seed presence, size and weight was recorded for each floret scored.

Key Results and Conclusions

The synchrony between pollen dehiscence and stigma collapse within a flower was dependent on its relative position in a spike and within a floret. Determined on the basis of synchrony within each flower, the level of pollination by pollen originating from other flowers reached approx. 30 % and did not change throughout the duration of flowering. A modelling exercise parameterized by flowering observations indicated that the temporal and spatial variability of anther activity within and between spikes may influence the relative resilience of wheat to sudden, extreme climatic events which has direct relevance to predicted future climate scenarios in the UK.     

云南元江干热河谷五种优势植物的内生真菌多样性

从蔓草虫豆（Atylosia scarabaeoides）、余甘子（Phyllanthus emblica）和黄花稔（Sida acuta）等5种云南元江干热河谷植物的525个组织块中,共分离得到内生真菌371株,内生真菌的分离频率在0.61～0.92之间,且所有植物叶内生真菌的分离频率都明显高于茎（P<0.05）。经形态学鉴定,内生真菌分属于拟茎点霉属（Phomopsis sp.）、离蠕孢属（Bipolaris sp.）和交链孢属（Alternaria sp.）等32个分类单元。拟茎点霉属为干热河谷植物优势内生真菌属,从所有被调查植物的茎叶中都分离得到该属真菌,且相对分离频率高达12.90%～50.54%。内生真菌群落组成的多样性和相似性分析结果表明,云南元江干热河谷植物内生真菌多样性偏低、宿主专一性较小。     

The DROOPING LEAF and OsETTIN2 genes promote awn development in rice

The awn is a long needle‐like appendage that, in some grass species, is formed on the lemma that encloses floral organs together with the palea. In rice, most wild species and most strains of Oryza sativa ssp. indica generate an awn, whereas most strains of O. sativa ssp. japonica do not. In japonica, the long‐awn characteristic appears to have been lost during domestication and breeding programs. Here, we found that the genes DROOPING LEAF (DL) and OsETTIN2 (OsETT2) are involved in awn development in the awned indica strain Kasalath. Genetic analyses and RNA‐silencing experiments indicate that DL and OsETT2 act independently in awn formation, and that either gene alone is not sufficient for awn development. Scanning electron microscopy revealed that the top region of the lemma (a putative awn primordium) is larger in an awned floret than in an awnless floret. OsETT2 is expressed in the awn primordium in the awned indica floret, but not in the awnless japonica floret except in the provascular bundle. DL is expressed underneath the primordium at similar levels in both indica and japonica florets, suggesting non‐cell‐autonomous action. We hypothesize that loss of expression of OsETT2 in the awn primordium is probably associated with the failure of awn formation in japonica strains.     

Identification of a novel homolog for a calmodulin-binding protein that is upregulated in alloplasmic wheat showing pistillody

Intracellular signaling pathways between the mitochondria and the nucleus are important in both normal and abnormal development in plants. The homeotic transformation of stamens into pistil-like structures (a phenomenon termed pistillody) in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) has been suggested to be induced by mitochondrial retrograde signaling, one of the forms of intracellular communication. We showed previously that the mitochondrial gene orf260 could alter the expression of nuclear class B MADS-box genes to induce pistillody. To elucidate the interactions between orf260 and nuclear homeotic genes, we performed a microarray analysis to compare gene expression patterns in the young spikes of a pistillody line and a normal line. We identified five genes that showed higher expression levels in the pistillody line. Quantitative expression analysis using real-time PCR indicated that among these five genes, Wheat Calmodulin-Binding Protein 1 (WCBP1) was significantly upregulated in young spikes of the pistillody line. The amino acid sequence of WCBP1 was predicted from the full-length cDNA sequence and found to encode a novel plant calmodulin-binding protein. RT-PCR analysis indicated that WCBP1 was preferentially expressed in young spikes at an early stage and decreased during spike maturation, indicating that it was associated with spikelet/floret development. Furthermore, in situ hybridization analysis suggested that WCBP1 was highly expressed in the pistil-like stamens at early to late developmental stages. These results indicate that WCBP1 plays a role in formation and development of pistil-like stamens induced by mitochondrial retrograde signaling.