A thaumatin-like effector protein suppresses the rust resistance of wheat and promotes the pathogenicity of Puccinia triticina by targeting TaRCA.

Thaumatin-like proteins (TLPs) in plants play a crucial role in combating stress, and they have been proven to possess antifungal properties. However, the role of TLPs in pathogens has not been reported. We identified a effector protein, Pt9029, which contained a Thaumatin domain in Puccinia triticina (Pt), possessing a chloroplast transit peptide and localized in the chloroplasts. Silencing Pt9029 in the Pt physiological race THTT resulted in a notable reduction in virulence and stunted growth and development of Pt hypha in near-isogenic wheat line TcLr2b. Overexpression of Pt9029 in wheat exerted a suppressive effect on H2O2 production, consequently impeding the wheat's disease resistance mechanisms. The TLP domain of Pt9029 targets the Rubisco activase (TaRCA) in chloroplasts. This interaction effectively inhibited the function of TaRCA, subsequently leading to a decrease in Rubisco enzyme activity. Therefore, this indicates that TLPs in Pt can inhibit host defense mechanisms during the pathogenic process of Pt. Moreover, TaRCA silencing resulted in reduced resistance of TcLr2b against Pt race THTT. This clearly demonstrated that TaRCA positively regulates wheat resistance to leaf rust. These findings reveal a novel strategy exploited by Pt to manipulate wheat rust resistance and promote pathogenicity.

Effector Pt9226 from Puccinia triticina Presents a Virulence Role in Wheat Line TcLr15.

Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of Puccinia triticina (Pt) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six Pt pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the Pt pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of Pt9226 in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of Pseudomonas fluorescens EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of PtEF-1α was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the Pt pathotype THTT was inoculated on a transient expression of Pt9226 in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the Pt pathotype THTT and wheat.

Wheat Leaf Rust Fungus Effector Protein Pt1641 Is Avirulent to TcLr1.

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H2O2 production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from ";" to "3", indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes.

The Underexplored Mechanisms of Wheat Resistance to Leaf Rust.

Wheat leaf rust, caused by the obligate biotrophic fungus Puccinia triticina Eriks. (Pt), is one of the most common wheat foliar diseases that continuously threatens global wheat production. Currently, the approaches used to mitigate pathogen infestation include the application of fungicides and the deployment of resistance genes or cultivars. However, the continuous deployment of selected resistant varieties causes host selection pressures that drive Pt evolution and promote the incessant emergence of new virulent races, resulting in the demise of wheat-resistant cultivars after several years of planting. Intriguingly, diploid wheat accessions were found to confer haustorium formation-based resistance to leaf rust, which involves prehaustorial and posthaustorial resistance mechanisms. The prehaustorial resistance in the interaction between einkorn and wheat leaf rust is not influenced by specific races of the pathogen. The induced defense mechanism, known as systemic acquired resistance, also confers durable resistance against a wide array of pathogens. This review summarizes the host range, pathogenic profile, and evolutionary basis of Pt; the molecular basis underlying wheat-Pt interactions; the cloning and characterization of wheat leaf rust resistance genes; prehaustorial and posthaustorial resistance; systemic acquired resistance; and the role of reactive oxygen species. The interplay between climatic factors, genetic features, planting dates, and disease dynamics in imparting resistance is also discussed.

The molecular structure, biological roles, and inhibition of plant pathogenic fungal chitin deacetylases.

Chitin/polysaccharide deacetylases belong to the carbohydrate esterases family 4 (CE4 enzymes). They play a crucial role in modifying the physiochemical characteristics of structural polysaccharides and are also involved in a wide range of biological processes such as fungal autolysis, spore formation, cell wall formation and integrity, and germling adhesion. These enzymes are mostly common in fungi, marine bacteria, and a limited number of insects. They facilitate the deacetylation of chitin which is a structural biopolymer that is abundantly found in fungal cell walls and spores and also in the cuticle and peritrophic matrices of insects. The deacetylases exhibit specificity towards a substrate containing a sequence of four GlcNAc units, with one of these units being subjected to deacetylation. Chitin deacetylation results in the formation of chitosan, which is a poor substrate for host plant chitinases, therefore it can suppress the host immune response triggered by fungal pathogens and enhance pathogen virulence and colonization. This review discusses plant pathogenic fungal chitin/polysaccharide deacetylases including their structure, substrate specificity, biological roles and some recently discovered chitin deacetylase inhibitors that can help to mitigate plant fungal diseases. This review provides fundamental knowledge that will undoubtedly lead to the rational design of novel inhibitors that target pathogenic fungal chitin deacetylases, which will also aid in the management of plant diseases, thereby safeguarding global food security.

Harnessing genetic resistance to rusts in wheat and integrated rust management methods to develop more durable resistant cultivars.

Wheat is one of the most important staple foods on earth. Leaf rust, stem rust and stripe rust, caused by Puccini triticina, Puccinia f. sp. graminis and Puccinia f. sp. striiformis, respectively, continue to threaten wheat production worldwide. Utilization of resistant cultivars is the most effective and chemical-free strategy to control rust diseases. Convectional and molecular biology techniques identified more than 200 resistance genes and their associated markers from common wheat and wheat wild relatives, which can be used by breeders in resistance breeding programmes. However, there is continuous emergence of new races of rust pathogens with novel degrees of virulence, thus rendering wheat resistance genes ineffective. An integration of genomic selection, genome editing, molecular breeding and marker-assisted selection, and phenotypic evaluations is required in developing high quality wheat varieties with resistance to multiple pathogens. Although host genotype resistance and application of fungicides are the most generally utilized approaches for controlling wheat rusts, effective agronomic methods are required to reduce disease management costs and increase wheat production sustainability. This review gives a critical overview of the current knowledge of rust resistance, particularly race-specific and non-race specific resistance, the role of pathogenesis-related proteins, non-coding RNAs, and transcription factors in rust resistance, and the molecular basis of interactions between wheat and rust pathogens. It will also discuss the new advances on how integrated rust management methods can assist in developing more durable resistant cultivars in these pathosystems.

The haustorium: The root of biotrophic fungal pathogens.

Biotrophic plant pathogenic fungi are among the dreadful pathogens that continuously threaten the production of economically important crops. The interaction of biotrophic fungal pathogens with their hosts necessitates the development of unique infection mechanisms and involvement of various virulence-associated components. Biotrophic plant pathogenic fungi have an exceptional lifestyle that supports nutrient acquisition from cells of a living host and are fully dependent on the host for successful completion of their life cycle. The haustorium, a specialized infection structure, is the key organ for biotrophic fungal pathogens. The haustorium is not only essential in the uptake of nutrients without killing the host, but also in the secretion and delivery of effectors into the host cells to manipulate host immune system and defense responses and reprogram the metabolic flow of the host. Although there is a number of unanswered questions in this area yet, results from various studies indicate that the haustorium is the root of biotrophic fungal pathogens. This review provides an overview of current knowledge of the haustorium, its structure, composition, and functions, which includes the most recent haustorial transcriptome studies.

Infection Strategies and Pathogenicity of Biotrophic Plant Fungal Pathogens.

Biotrophic plant pathogenic fungi are widely distributed and are among the most damaging pathogenic organisms of agriculturally important crops responsible for significant losses in quality and yield. However, the pathogenesis of obligate parasitic pathogenic microorganisms is still under investigation because they cannot reproduce and complete their life cycle on an artificial medium. The successful lifestyle of biotrophic fungal pathogens depends on their ability to secrete effector proteins to manipulate or evade plant defense response. By integrating genomics, transcriptomics, and effectoromics, insights into how the adaptation of biotrophic plant fungal pathogens adapt to their host populations can be gained. Efficient tools to decipher the precise molecular mechanisms of rust-plant interactions, and standardized routines in genomics and functional pipelines have been established and will pave the way for comparative studies. Deciphering fungal pathogenesis not only allows us to better understand how fungal pathogens infect host plants but also provides valuable information for plant diseases control, including new strategies to prevent, delay, or inhibit fungal development. Our review provides a comprehensive overview of the efforts that have been made to decipher the effector proteins of biotrophic fungal pathogens and demonstrates how rapidly research in the field of obligate biotrophy has progressed.

Fungal Secondary Metabolites and Small RNAs Enhance Pathogenicity during Plant-Fungal Pathogen Interactions.

Fungal plant pathogens use proteinaceous effectors as well as newly identified secondary metabolites (SMs) and small non-coding RNA (sRNA) effectors to manipulate the host plant's defense system via diverse plant cell compartments, distinct organelles, and many host genes. However, most molecular studies of plant-fungal interactions have focused on secreted effector proteins without exploring the possibly equivalent functions performed by fungal (SMs) and sRNAs, which are collectively known as "non-proteinaceous effectors". Fungal SMs have been shown to be generated throughout the plant colonization process, particularly in the early biotrophic stages of infection. The fungal repertoire of non-proteinaceous effectors has been broadened by the discovery of fungal sRNAs that specifically target plant genes involved in resistance and defense responses. Many RNAs, particularly sRNAs involved in gene silencing, have been shown to transmit bidirectionally between fungal pathogens and their hosts. However, there are no clear functional approaches to study the role of these SM and sRNA effectors. Undoubtedly, fungal SM and sRNA effectors are now a treasured land to seek. Therefore, understanding the role of fungal SM and sRNA effectors may provide insights into the infection process and identification of the interacting host genes that are targeted by these effectors. This review discusses the role of fungal SMs and sRNAs during plant-fungal interactions. It will also focus on the translocation of sRNA effectors across kingdoms, the application of cross-kingdom RNA interference in managing plant diseases and the tools that can be used to predict and study these non-proteinaceous effectors.

Wheat leaf rust fungus effector Pt13024 is avirulent to TcLr30.

Wheat leaf rust, caused by Puccinia triticina Eriks. (Pt), is a global wheat disease threatening wheat production. Dissecting how Pt effector proteins interact with wheat has great significance in understanding the pathogenicity mechanisms of Pt. In the study, the cDNA of Pt 13-5-72 interacting with susceptible cultivar Thatcher was used as template to amplify Pt13024 gene. The expression pattern and structure of Pt13024 were analyzed by qRT-PCR and online softwares. The secretion function of Pt13024 signal peptide was verified by the yeast system. Subcellular localization of Pt13024 was analyzed using transient expression on Nicotiana benthamiana. The verification that Pt13024 inhibited programmed cell death (PCD) was conducted on N. benthamiana and wheat. The deletion mutation of Pt13024 was used to identify the virulence function motif. The transient transformation of wheat mediated by the type III secretion system (TTSS) was used to analyze the activity of regulating the host defense response of Pt13024. Pt13024 gene silencing was performed by host-induced gene silencing (HIGS). The results showed that Pt13024 was identified as an effector and localized in the cytoplasm and nucleus on the N. benthamiana. It can inhibit PCD induced by the Bcl-2-associated X protein (BAX) from mice and infestans 1 (INF1) from Phytophthora infestans on N. benthamiana, and it can also inhibit PCD induced by DC3000 on wheat. The amino acids 22 to 41 at N-terminal of the Pt13024 are essential for the inhibition of programmed cell death (PCD) induced by BAX. The accumulation of reactive oxygen species and deposition of callose in near-isogenic line TcLr30, which is in Thatcher background with Lr30, induced by Pt13024 was higher than that in 41 wheat leaf rust-resistant near-isogenic lines (monogenic lines) with different resistance genes and Thatcher. Silencing of Pt13024 reduced the leaf rust resistance of Lr30 during the interaction between Pt and TcLr30. We can conclude that Pt13024 is avirulent to TcLr30 when Pt interacts with TcLr30. These findings lay the foundation for further investigations into the role of Pt effector proteins in pathogenesis and their regulatory mechanisms.

Combating powdery mildew: Advances in molecular interactions between Blumeria graminis f. sp. tritici and wheat.

Wheat powdery mildew caused by a biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a widespread airborne disease which continues to threaten global wheat production. One of the most chemical-free and cost-effective approaches for the management of wheat powdery mildew is the exploitation of resistant cultivars. Accumulating evidence has reported that more than 100 powdery mildew resistance genes or alleles mapping to 63 different loci (Pm1-Pm68) have been identified from common wheat and its wild relatives, and only a few of them have been cloned so far. However, continuous emergence of new pathogen races with novel degrees of virulence renders wheat resistance genes ineffective. An essential breeding strategy for achieving more durable resistance is the pyramiding of resistance genes into a single genotype. The genetics of host-pathogen interactions integrated with temperature conditions and the interaction between resistance genes and their corresponding pathogen a virulence genes or other resistance genes within the wheat genome determine the expression of resistance genes. Considerable progress has been made in revealing Bgt pathogenesis mechanisms, identification of resistance genes and breeding of wheat powdery mildew resistant cultivars. A detailed understanding of the molecular interactions between wheat and Bgt will facilitate the development of novel and effective approaches for controlling powdery mildew. This review gives a succinct overview of the molecular basis of interactions between wheat and Bgt, and wheat defense mechanisms against Bgt infection. It will also unleash the unsung roles of epigenetic processes, autophagy and silicon in wheat resistance to Bgt.