Temporal behavior of wheat – Puccinia striiformis interaction prompted defense-responsive genes

ABSTRACT Wheat stripe rust caused by Puccinia striiformis Westend. f.sp. tritici (Pst) is a global threat to wheat production. Genetic modification of defense-responsive factors in wheat rust interactions could help devise strategies to control stripe rust on wheat. This experiment studied the interaction between Pst pathotype 78S84 in PBW343 and FLW-3 by evaluating the quantitative temporal transcription profiles of defense-related genes at different time points. This is the first attempt to exhibit inter-connections among different proteins and depict a hypothetical model for the mechanism of R gene-mediated resistance. Transcript levels of LTP, AQP1, PR1, PR2, PR4, and PR10 were relatively higher under compatible interaction, while under incompatible interaction, transcript levels of COMT1, PRA2, WCAB, and PR9 were significantly high. This study projected the role of defense-responsive genes, inter-networking of proteins, and R gene-mediated resistance between wheat and stripe rust.


Introduction
Wheat (Triticum aestivum L.) is a significant component of the food chain, along with rice and pulses. It has provided nutrition to the world population over the centuries, contributing around 20% calories of the total food intake. Although wheat has reached yield plateau, its demand is still increasing with ever-increasing global population. However, due to the rapidly changing climate and genetic homogeneity, the wheat crop is exposed to various biotic and abiotic stresses, which create various levels of threats to yield, impacting its production considerably (Lata et al. 2021).
Stripe (Yellow) rust of wheat is one of the major diseases caused by Puccinia striiformis Westend. f.sp. tritici (Pst) mainly in cooler cropping areas (Gangwar et al. 2016). This disease sometimes leads to complete crop failure if not appropriately treated (Hovmøller et al. 2010). Stripe rust pathotype 78S84 was isolated from PBW343 grown in the Batala area of Punjab, India. It is one of the most aggressive pathotypes and has been introduced from Western Europe or Eastern Africa to South Asia and India (Gangwar et al. 2022;Prashar et al. 2007Prashar et al. , 2015. Being an obligate pathogen, Pst is entirely dependent on its host for survival. The infection starts when aerial spores settle on leaf/green tissues and invade through the stomata. The spores further proliferate by hyphal extension, which produces haustoria as a feeding organ and forms intimate connections with plant tissues for nutrient uptake. Breeding for stripe rust using resistant germplasm is an effective and eco-friendly approach (Dubin and Brennan 2009;Singh et al. 2016). Approximately 83Yr genes and several quantitative trait loci (QTLs) associated with stripe rust resistance have been identified in wheat to date (Wang et al. 2021). However, the quick evolution of virulent races on varieties with single gene governed resistance leads to the loosing of resistance genes. Pyramiding genes and QTLs from diverse sources is a tiresome process and is often associated with linkage drag. Therefore, to develop broad-spectrum and durable rust resistance, novel strategies are required. A complete understanding of the molecular basis of plant-pathogen interaction will be the first step. Nevertheless, the molecular basis of defense pathways involved in wheat and stripe rust pathogen interactions is yet to be uncovered. Host-pathogen interaction is the first event for infection, and exploring the host-pathogen interface is a key to uncovering the molecular mechanisms that control disease progression.
Plant resistance against a pathogen is regulated by the genetic actions of the host and pathogen. This is a type of molecular armed race termed as 'Z scheme.' Plants recognize specific molecular signals/patterns and initiate a cascade of strong defense-responsive genes. The regulation of defenseresponsive genes governs effective immune response under pathogen attack, initiated quickly and, therefore, R genemediated resistance has shown effective results (Prasad et al. 2019). This regulation decides the fate of the battle between host and pathogen, expressed as the compatible and incompatible response (Jones and Dangl 2006). Hydrolytic enzymes (e.g. chitinases and β-1,3-glucanases), peroxidases, aquaporins, caffeic acid O-methyltransferase, chlorophyll a/b-binding proteins, Type 1 non-specific lipid transfer protein precursor, phytoalexin biosynthetic enzymes, and glutathione S-transferases are few key defense-related proteins produced in response to pathogen attack (Ebrahim and Singh 2011).
Nevertheless, Pst is a devastating wheat pathogen which causes significant yield loss. A little information is available molecularly, specifically during the compatible and incompatible interactions between wheat and stripe rust pathogen. Therefore, in the present experiment, expression profiling of candidate defense-responsive genes was conducted in PBW343 and FLW-3 wheat stocks during stripe rust infection. In addition, we tried to uncover interconnections among other proteins and depicted a hypothetical model of R gene-mediated resistance in wheat against yellow rust for the first time. Expression of these genes under pathogenic stress in wheat cultivars showing contrasting responses might be helpful to speculate on the possible role and understand the molecular mechanisms of resistance and susceptibility governed by these genes.

Plant material and pathogen
The wheat stocks PBW343 and FLW-3 were used as experimental plant materials, which show susceptibility and resistance to the pathotype 78S84 of Pst, respectively. This was one of the most predominant and devastating races of stripe rust, virulent on Yr9 + Yr27 present in PBW343 (cultivated over a large area of India). Airulence⁄virulence formula of pathotype 78S84 is mentioned in supplementary file 1. Single spore derived culture maintained on the seedlings of a susceptible durum wheat cultivar A9-30-1 at ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Regional Station, Flowerdale, Shimla, India, was used in the study. A9-30-1 was grown in aluminum bread pans/trays (29 × 12 × 7 cm) filled with a mixture of autoclaved composite soil (peat, sand, and soil in 1:1:1 ratio) inside the growth chamber under controlled temperature conditions (28°C/ 22°C), relative humidity (80%), and photoperiod (14 h day with 300 lux light; 10 h dark periods).

Inoculation, disease assessment, and sample collection
Freshly collected uredospores of 78S84 pathotype were used to prepare inoculum by suspending non-phytotoxic isoparaffinic mineral oil Soltrol (Chevron Phillips Chemical Company, US). Two-week-old seedlings of PBW343 and FLW-3 with fully expanded flag leaves were spray inoculated. In control, wheat seedlings were sprayed with mineral oil only. After inoculation, seedlings were sprayed with a fine mist of water and were placed overnight under high humidity (>90%) in dew chambers to get infection and then shifted to the normal growth conditions in a greenhouse maintained at 16 ± 2°C with 60-80% relative humidity and ∼15,000 lx requisite intensity of light for 12 h was maintained. After two weeks post-inoculation, infection types (ITs) were scored according to Stakman's scale (Stakman et al. 1962) with some modifications (Roelfs 1984;Roelfs et al. 1992). For RNA isolation, leaf samples from all the test plants were harvested at 0, 1, 3, 6, 12, 24, and 48 h post-inoculation (hpi) and frozen immediately in liquid nitrogen, stored at −80°C until RNA isolation. After 15 days, post-inoculation (dpi) infection was confirmed by the visual examination of the remaining wheat plants in trays.

Total RNA isolation and cDNA preparation
Total RNA was extracted from flag leaf tissue (100 mg) of all the samples in three biological replicates using the QIAGEN RNA (Germany, Maryland) isolation kit as per the manufacturer's protocols. The quantity and purity of extracted RNA were analyzed using a NanoDrop 2000c® UV-Vis Spectrophotometer (Thermo Scientific). The RNA isolated from the samples of three biological replicates of individual treatments was pooled for each time point. Two µg total RNA from all the treatments at different time points was used to synthesize cDNA using High-Capacity cDNA Reverse Transcription Kit with Oligo(dT) primer (Applied Biosystems).

Selection of reference and candidate PR genes
Full-length mRNA coding sequences of selected defenseresponsive and house-keeping genes specific to wheat were obtained from the National Center for Biotechnology Information (NCBI) database. The most conserved region of 10 defense-responsive genes i.e., caffeic acid O-methyltransferase (COMT1), class III peroxidase (PRA2), Type 1 nonspecific lipid transfer protein precursor (LTP1), chlorophyll a/b-binding protein WCAB precursor (WCAB), aquaporin (AQP1), β-1,3-glucanase (PR1 and PR2), endochitinase (PR4), peroxidase (PR9) and phenylalanine ammonia-lyase (PR10), along with three house-keeping genes were utilized in the current study (Table 1). For endogenous control, three genes, viz.; Ubiquitin (UBI), elongation factor-1α (EF-1 α), and Glucose-6-phosphate dehydrogenase (G 6 DPH), were selected and tested. Primer3Plus program was used to design the primers for qRT-PCR analysis. Norm-Finder (Andersen et al. 2004; http://www.multid.se/genex/ hs410.htm) and Best Keeper (Pfaffl et al. 2004) software were used to analyze the best house-keeping gene for relative quantification analysis, which is crucial for precise normalization of gene expression analysis (Savadi et al. 2018;Prasad et al. 2019). Among these three housekeeping genes, EF-1 α was found the most stable housekeeping gene by both methods based on the least stability value (0.008) in Norm-Finder and maximum coefficient of correlation (0.905) in Best Keeper analysis (Table 2).

Amplification efficiency and specificity of genes
Ten fold dilutions of the cDNA template in the qRT-PCR were used to determine the amplification efficiency of each primer pair, which ranged 95-103%, indicating optimal reaction conditions for exponential amplification of the target gene. Melting curve analysis (McGrann et al. 2009) was performed to check the specificity of each primer pair and confirmed through agarose gel electrophoresis of qRT-PCR amplicons.

qRT-PCR expressional analysis
Three replicates of cDNA from all the samples were amplified using gene-specific primer using FG-Power SYBR Green PCR Master Mix (Applied Biosystems) for qRT-PCR analysis carried out in CFX96 Real-time Sys-tem1000 Touch thermal cycler (BIO-RAD). The reaction was carried out at initial activation at 95°C for 7 min, followed by 40 cycles of 10 s at 95°C and 30s at 60°C. It was heated with a rate of 0.1°C per second starting at 60°C to 95°C to melt to check the specificity of the primers. The total 20 μL reaction mixture contained 10 μL Power SYBR Green PCR Master Mix, 1 μL cDNA, and 0.5 μL of each primer at a final concentration of 5 μM was taken to amplify cDNA. CFX Maestro Software was used for instrument operation, data collection, analysis, and graphing of qRT-PCR data. At each polymerization point, fluorescence signals were collected, and a threshold constant value (Ct) was calculated from the amplification curve by selecting the optimal ΔRn (Rieu and Powers 2009). According to Paolacci et al. (2009), candidate gene expression levels were computed relative to the EF-1 α expression (best performing housekeeping reference gene) at the identical time points using the 2 −ΔΔCT .

STRING analysis for defense-responsive genes
A functional protein association network was developed for defense-responsive genes through STRING database v10 (Szklarczyk et al. 2015). This analysis helps tfind the important proteins to which defense-regulated proteins may interact.

Statistical analysis
To determine significantly different time points for temporal expression of defense-responsive genes, one-way analysis of variance (ANOVA) and Tukey's post hoc tests are performed at P ≤ 0.05.

Result
The sessile nature of plants makes them more prone to several biotic stresses, which they have to evade by evolving defense systems against pathgogen. As each pathogen uses a different strategy for plant infection, plants have to come up with specific counteract. In the case of wheat rust infection cascade of various defense-responsive genes started to guard against the damage caused by the pathogen. This experiment uncovers expression regulation of defenseresponsive genes at different time points during Pst infection on wheat. In the present study, an attempt was made to elucidate the behavior of various defense genes in wheat cultivars showing differential responses during stripe rust infection. Phenotypic observations of disease symptoms The seedlings of PBW343 and FLW-3 growing in control and treatment were observed regularly for twenty days of postinoculation to confirm susceptibility or resistance responses. Disease symptoms developed 5th dpi onward on PBW343 while flecking occurred 7th dpi on FLW-3 and was finally recorded on 15th dpi. Cultivar FLW-3 gave ;to ; type of responses, while PBW343 showed 33+ to 4 types of reactions ( Figure 1). The post-inoculation response of wheat lines reconfirms the susceptibility and resistance of PBW343 and FLW-3 to pathotype 78S84, respectively. On the other hand, mock-inoculated seedlings did not exhibit any symptoms related to resistance /susceptibility. The phenotypic screening was also conducted in replicate under a similar set of conditions to confirm the results.

Differential gene expression during compatible and incompatible interactions
Differential expressions of ten defense-responsive genes (COMT1, PRA2, LTP1, WCAB, AQP1, PR1, PR2, PR4, PR9, and PR10) were analysed at different time points (0, 1, 3, 6, 12, 24, and 48hpi) to understand the expression pattern of these genes during incompatible and compatible reactions among stripe rust pathogen and wheat accessions. After inoculation, pathogen and host under-go a cascade of signaling to overcome each other's strategy. In this experiment result of RT qPCR of the candidate defense, responsive genes show substantial changes in expression profile at different time courses. Expression analysis of the studied genes showed contrasting behavior under compatible and incompatible interactions. Under compatible interactions of wheat and stripe rust, the AQP1 gene expressed higher throughout the time points, whereas WCAB, PR9, and PR10 showed a moderate level of expression. There were fewer expressions of COMT1, PRA2, LTP1, PR1, PR2, and PR4. On the other hand, the incompatible interaction, WCAB and PR9 was found to be highest, while COMT1, PRA2, LTP, AQP, and PR10 were moderately expressed. Lower expression levels were recorded for genes PR1, PR2, and PR4 (Figure 2(A,B)). In our study expression of COMT1 varied from 1.19 to 8.22 fold in compatible interactions compared to 3.13-5.22 fold under incompatible interactions. Although COMT1 expressed relatively higher under incompatible interaction for the whole time course, the maximum expression was found at 3hpi in compatible and incompatible interactions. PRA2 showed significantly constant expression till 3hpi and increased at 6hpi; further, its expression reached at the highest level on 48 th hpi after reduction at 12 and 24hpi in PBW343 (Figure 3(B)). The expression levels of LTP1 started from 1.33 fold at 3hpi under incompatible interactions and reached at a higher level at 12hpi up to 15.73 fold. On the other hand, in compatible interaction, the lowest expression was found at 3hpi i.e., 0.99-fold, and the highest expression level was recorded at 24hpi i.e., 11.87-fold (Figure 3(C)). There was an exponential up-regulation for the WCAB gene at most of the time points apart from 0hpi (1.05 fold)   to 3hpi (6.23 fold) under incompatible interaction. Antagonistically in susceptible wheat, the cultivar expression level of WCAB was recorded lowest at 12 and 24hpi while maximum at 3hpi (47.83 fold) (Figure 3(D)). A strong contradictory behavior was found in the expression of the AQP1 gene among compatible and incompatible interactions (Figure 3 (E)). Under compatible interaction, AQP1 expression level was higher up to 80 folds at initial time points and then reduced on 3 rd hpi, again increasing up to 24hpi but increasing sharply at 48hpi. However, under incompatible interaction, expression levels of AQP1 were lower than ten folds except 6 and 24hpi, i.e. 16.91 and 19.02 folds, respectively.
Expression levels of PR1 were lower than ten folds. Without significant differences in either of the wheat accessions except at 3hpi in PBW343 and FLW-3, the expression level of PR1 increased slightly to 6hpi but decreased again at later time points (Figure 3(F)). Expression levels of PR2 were also lower than ten folds without any significant modulations between cultivars, and maximum expression was found at 7.03 folds under compatible interaction at 1hpi and after that, decreased slightly (Figure 3(G)). The expression levels of PR4 in the compatible interaction started from 1.61 folds at 0hpi and increased significantly up to 3hpi (11.59 folds). Expression declined gradually in later time stages and decreased up to 1.77 folds on 48hpi. Under incompatible interaction expression level of PR4 swung for initial time points with a maximum at 1hpi (Figure 3(H)). The expression level of PR9 was significantly high under incompatible interaction at initial time points, then reduced gradually at 12 and 24hpi but increased sharply at 48hpi and was found to be maximum at this point (30.91 folds). Under compatible interaction, a different trend was found in expression levels of PR9 (Figure 3(I)). PR10 expression under compatible interaction started with 3.02 folds at 0hpi, then decreased at the next time point but increased exponentially on 3hpi (63.71 folds), which was the highest among either of the cultivars at each time point. A similar trend was followed under incompatible interaction up to 3hpi. After that expression pattern was different compared to compatible interaction. The lowest level of PR10 expression was found at 24hpi (1.85 folds) under incompatible interaction (Figure 3(J)).
For time points, a comparison of normalized expressions data of stress-responsive genes presented contrasting behavior under compatible and incompatible interaction. Under compatible interaction, gene expressions increased initially up to 3hpi, then declined significantly for the next two time points, and the lowest gene expression was reported on 12hpi. After this time point, gene expression started increasing for 24hpi and 48hpi. On the other hand, under incompatible interaction, an inverse trend was found for normalized expression data of stress-responsive genes. Initially, gene expression was minimum and gradually increased significantly from 0hpi to 12hpi and, after that, decreased slowly at 24hpi and 48hpi (Figure 4(A,B)).

STRING analysis of selected defense-responsive genes
These defense-responsive proteins showed an interaction network with other proteins working inside the plant cells. String analysis showed a set of connections of these proteins with supplementary proteins, which are involved in the inhibition of pathogens ( Figure 5). COMT1 interacts with Traes_6DS_211935E65.2 (CAD-Cinnamyl alcohol dehydrogenase), which is involved in the lignin biosynthetic process. At the time of infection, lignification of the cell walls induces defense signaling. PR10 (1AS_0B9295A68.1) is homologous of AT2G37040.1, found in Arabidopsis, interacted with cinnamoyl-CoA reductase, 4-coumarate: CoA ligase and caffeoyl-CoA O-methyltransferase, which participate in cell wall synthesis and strengthening. These proteins start a cascade to strengthen plant cell walls by lignin deposition and counteract the invasion of a pathogen. Traes_5BL_628390692.1 (LTP) showed interactions with Traes_5BL_9BE35DEC71.1, Traes_2BL_490540C70.1 and Traes_4BL_AB3EBF1FD.2. These proteins are implied in the transfer of lipids across the membrane and the binding of actin in the cytoskeleton. These proteins strengthen the cytoskeleton of cells and inhibit the entry of pathogens.

Discussion
Several defense-responsive genes are induced during pathogen attack, also known as R genes (Yu et al. 2010;Dmochowska-Boguta et al. 2015;Savadi et al. 2018;Zhang et al. 2018;Wang et al. 2020). Similarly, wheat has evolved several defense-related genes against Puccinia striiformis. Usually, the interaction between plants and pathogens may be compatible and incompatible depending on the recognition of the pathogen, signal transduction pathway activation, and initiation of active defense molecules. In the case of Pstwheat interaction, a series of events occur at a different time point after the pathogen's attack. Three hours postinoculation, urediospores germinate on the leaf surface to form an appressorial swelling over the stomatal opening. In the next 3 h, appressorial hyphae reach the sub-stomatal cavity and form a sub-stomatal vesicle. These hyphae form contacts with mesophyll cells and differentiate them into haustorial mother cells in around 12 h post-inoculation and then formes feeding structures in 24 h (Zhang and Dickinson 2001). Under incompatible interaction, pre-haustorial resistance plays an important role in ceasing the infection (Wesp-Guterres et al. 2013).
After a pathogen attack, the plant stimulates various signaling pathways and defense mechanisms such as cell wall modification, ROS generation, hydrolytic enzyme, hypersensitive response, and systemic acquired resistance (Singh et al. 2014). Expression and regulation of these genes at different stages of pathogen attack govern the responses of a host in terms of resistance and susceptibility. In the present study, an attempt was made to delineate the expression profiling of selected defense response genes during Pst-wheat interaction. Differential expression of these defense-responsive genes was observed at different time courses. Nevertheless, the results of this experiment supplement the role of varying defense-responsive genes under compatible and incompatible wheat interactions with stripe rust pathogen. R genes confer resistance under incompatible reactions, while S genes (susceptibility genes) are a crucial component of compatible interactions (Henningsen et al. 2021). COMT1 is a key enzyme that catalyzes a step in the biosynthesis of monolignols, precursors of essential plant cell wall component lignin. Lignin is covalently bonded to the cellulose and hemicellulose and regulates the movement of water and nutrients in addition to playing a significant role in plant defense (Vance et al. 1980;Tu et al. 2010). STRING analysis revealed the connection of COMT1 with cinnamyl alcohol dehydrogenase functioning in the lignin biosynthetic process. Lignification of cell walls significantly induces a defense cascade in wheat during stripe rust infection (Moldenhauer et al. 2008). In our results, the expression of COMT1 was relatively higher during infection under incompatible interactions and showed resistance in FLW3 to stripe rust. However, in compatible interactions, the expression of COMT1 was maximum at 3hpi and reduced afterward, which indicates its overexpression till the post-haustorial stage in the susceptible cultivar. Subsequently, PBW343 could not inhibit further pathogen invasion due to the weakening of the cell wall.
PRA2 is a member of the Class III peroxidases family, a class of enzymes involved in the reduction of peroxides and several physiological and developmental processes, such as cell wall formation, germination to senescence process, production of ROS, etc. (Kidwai et al. 2020). In the present experiment, induction in the expression level of this gene is significantly higher under incompatible interactions from 6hpi might help plant cells to mediate host resistance. In addition, it may restrict pathogen invasion before the formation of haustoria and restricting the spread of the pathogen through ROS-mediated resistance.
Type 1 non-specific lipid transfer protein precursors (LTPs) transport lipids or wax transversely through membranes to form mechanical barriers and are involved in plant defense due to antibiotic properties. The defense function of LTPs is associated with the transfer of signaling mediators and acting like endogenous elicitors in association with lipid molecules recognized by specific receptors and trigger an immune response (Finkina et al. 2016). This study showed the up-regulation of LTPs under incompatible interactions at the time of membrane damage as the transport of lipids would be essential for repairing damaged tissue. Conversely, the expression of LTPs was high in compatible interaction at the initial stage, which indicates that the susceptible cultivar also tries to defend itself against pathogen attack. Still, in later infection stages, the pathogen overcomes such barriers created by the host and results in susceptibility.
Chlorophyll a/b-binding protein WCAB precursors (WCAB) are apo-proteins and formed the light-harvesting complex of photosystem II in association with chlorophylls a and b. WCAB has a significant role in the energy generation process photosynthesis, as it is part of the photo-system II complex in the chloroplast. The chlorophyll a/b binding protein stabilizes the photosystem I and II by balancing excitation energy (Casassola et al. 2015). This experiment showed expression of WCAB was higher in resistant cultivar, which is supported through either of two mechanisms. The first one is ROS-mediated defense response signaling (Pospíšil 2012), and the second is high-sugar-mediated resistance (Morkunas et al. 2014). According to the first mechanism, excitation energy absorbed by chlorophylls is converted into the energy of separated charges consequent water-plastoquinone oxidoreductase activity. This is coupled with the formation of reactive oxygen species and is involved in the defense pathway. The second mechanism supports a high level of sugars in plant tissues and increases plant resistance in most of the fungal pathogen and plant systems. WCAB proteins are involved in sugar generation through photosynthesis, and this sugar acts as a primary substrate for providing energy and structural material for defense responses. Besides that, sugars act as signaling molecules and regulate Figure 6. Diagrammatic illustration of a hypothetical model for R gene-mediated resistance in wheat against yellow rust. Defense-responsive genes show diverse transcriptional regulations under compatible and incompatible interaction between wheat and stripe rust deliberated susceptibility and resistance response. (Solid and dotted arrows designate relatively higher and lower expression of defense-responsive genes) the plant immune system through several processes; cell wall lignification, synthesis of flavonoids, and induce certain PR proteins. Sugars enhance oxidative burst at the early stages of infection through the generation of molecular oxygen as a byproduct of the energy conversion process, which utilizes an ROS precursor and involved in the defense mechanism. Several sugars perform as priming agents, inducing resistance response toward pathogen (Lata et al. communicated). Nevertheless, in several studies, WCAB showed higher expression under compatible interaction, which could favor energy balance for defense-responsive genes (Prasad et al. 2019;Cho et al. 2006).
Aquaporins (AQP1) are integral membrane proteins that form pores in cell membranes; these proteins facilitate the flow of water faster than by diffusion across the phospholipid bilayer. AQP1 proteins form the channels for water movement and the selective flux of several small solutes involved in various essential processes crossways the membranes. AQPs are significant regulators of plant-pathogen interactions, leading to either plant immunity or pathogenicity (Li et al. 2020). Several studies confirmed the dual behavior of AQPs that AQPs could either be involved in susceptibility as they are constantly under the threat of being hijacked by pathogens for infecting the plants or can also be involved in plant resistance through the transportation of H 2 O 2 . Interactions between rice and the bacterial blight pathogen Xanthomonas oryzae pv. Oryzae, AQPs confer susceptibility of plant cells (Li et al. 2019), while in Arabidopsis (a non-host of Xoo), Hpa1 interacts with the plant H 2 O 2 transport channel AtPIP1;4 resulting in the H 2 O 2 -mediated defense (Tian et al. 2016). In this study, higher expression under compatible interactions suggested the induction of AQP1 expression, increasing pore formation. By this means the flow of water increases within cells to promote haustoria development. Similar results were revealed by Casassola et al. (2015).
Pathogenesis-related (PR) proteins are usually induced by the attack of several pathogens and defense-related signaling molecules (Oide et al. 2013). These are a group of diverse molecules and key components of systemic acquired resistance (SAR), which we can say plant innate immunity (Ali et al. 2018). PR1is a β -1, 3-glucanase enzyme coding gene involved in the degradation of fungal cell walls, and required for the hydrolysis of glucan present in the cell wall. Besides, those PR-1 genes were induced through several signal transduction pathways, such as salicylic acid, jasmonic acid, and/ or ethylene in tobacco. PR-2 proteins are also categorized as β-1,3-glucanases having 1,3-β-endoglucanase activity. These enzymes catalyze the hydrolytic cleavage of 1,3-β-D-glucosidic bonds of β-1,3-glucans for the weakening of the cell wall of a pathogen (Singh et al. 2014). In the present experiment, both enzymes showed continuous expression through all time points.
PR4 are endo-chitinase coding genes that degrade chitins present in fungal cell walls, so these enzymes inhibit the growth of the pathogen by impeding the haustorium mother cell formation and spreading secondary infection. Our results showed the up-regulation of PR4 in the initial stages under incompatible interactions. PR4 proteins are considered the signature genes under the jasmonic acid pathway in models and many crop plants (Ali et al. 2018). PR9 proteins played a significant role in ROS generation and mechanical barrier formation to prevent the pathogen's spread and by degrading the pathogens. Higher expression of PR9 under incompatible interactions at later time points showed the restricted growth and death of the pathogen.
PR10 genes encode phenylalanine ammonia-lyase (PAL) and are involved in the biosynthesis of flavonoids, phenylpropanoids, and lignins by the phenyl propanoid pathway (Casassola et al. 2015), and showed ribonuclease activity (Filipenko et al. 2013;Besbes et al. 2019). STRING analysis showed the networking of PR10 with cinnamoyl-CoA reductase, 4-coumarate: CoA ligase, and caffeoyl-CoA Omethyltransferase. These proteins are involved in the lignifications of the cell wall. Lignin biosynthesis extensively contributes to various biotic and abiotic stresses (Liu et al. 2018). Under compatible interactions, PR10 expression was increased exponentially at the initial stage but lacked the constant expression and failed to prevent the fungal growth. String analysis showed connections of these defense-responsive proteins with other proteins. The networking of proteins initiates the cascade of signals to stop pathogen entry. Orthologous proteins from other plants revealed inhibiting actions against pathogen invasion (Liu et al. 2018;Fleury et al. 2019). Defense-responsive genes studied in the present experiment showed differential gene expression under compatible and incompatible interactions of Puccinia striiformis and wheat. Although the real-time expression data provide important clues about the participation of these genes in the specific host-pathogen interactions, extensive reverse genetic-base characterization of these genes is needed to conclude about their role under plant-pathogen interactions.
Based on temporal transcriptional changes of defenseresponsive genes, the authors propose a hypothetical model for the mechanism of R gene-mediated resistance against yellow rust, depicted in Figure 6. Under incompatible interactions between FLW3 and 78S84, Avr gene products (effector proteins) are released by the pathogen and are recognized by the host-resistance (R) gene product present in the host cell. R genes encode nucleotide-binding and leucine-rich repeat (NLR) proteins. During infection, these NLR proteins act as immune receptors and recognize pathogen effector proteins delivered into host cells. The aforementioned interactions among effector proteins and NLR proteins cause an effector-triggered immunity (ETI). Many defense-responsive genes are up-regulated (COMT1, PRA2, WCAB, and PR9) in response to the signaling cascade and activate innate immunity to induce host resistance. Up-regulation of these genes activates several defense pathways, viz. plant cell wall synthesis and repair, peroxidases, H 2 O 2induced hypersensitive reaction, energy generations for metabolic processes, ROS generation, and mechanical barrier formation to prevent pathogen spread. Contrastingly, under compatible interactions between PBW343 and 78S84, the pathogen avirulence (Avr) gene product has failed to recognize the host-resistance (R) gene product or receptor protein in the host cell. In the absence of pathogen recognition, a different secondary messenger's induction cascade takes place, which modulates the physiology of the host cell. These secondary messengers activate some transcription factors, which penetrate in the nucleus. These factors regulate the transcription of different genes. AQP1, LTP, PR1, PR2, PR4, and PR10 genes showed up-regulation and helped in water supply for haustoria formation, development, and spread of the pathogen from one cell to another. The down-regulation of COMT1, PRA2, WCAB, and PR9 genes suppresses H 2 O 2 -induced hypersensitive response and promotes infection/ susceptibility.

Conclusion
Stripe rust is a most devastating fungal disease of wheat and causes significant crop loss in many wheat-growing countries. Transcriptional gene expression analysis of defense-responsive genes indicated the differential regulations under compatible and incompatible interactions at different time points. We conclude from expression analyses that COMT1, PRA2, WCAB, and PR9 played an essential role under incompatible interactions, while LTP, AQP1, PR1, PR2, PR4, and PR10 played a significant role in compatible interactions. Gene expression induced after infection of Pst pathotype at initial stages in both the conditions. However, the overall expression of defense-responsive genes was higher at 6-24hpi under compatible interaction that showed the pre-haustorial resistance and a potential PAMP-Triggered-Immunity (PTI) response. The up-regulation of defense-responsive genes from 3hpi to 24hpi prevents the growth of appressorial hyphae, haustorial mother cell development, and the formation of feeding structures. At later stages, post-haustorial (hypersensitive) resistance was provided by COMT1, PRA2, WCAB, and PR9 genes, which showed higher transcription levels at 24hpi to 48hpi might be a sign of Effector-Triggered-Immunity (ETI) response. The results of this experiment elucidate the action of defense-responsive genes at the molecular level under compatible and incompatible interactions.