Stripe Rust Effector Pst_9302 Inhibits Wheat Immunity to Promote Susceptibility

Puccinia striiformis f. sp. tritici is an obligate biotrophic fungus that causes destructive stripe rust disease in wheat. During infection, Pst secretes virulence effectors via a specific infection structure—the haustorium—inside host cells to disturb host immunity and promote fungal colonization and expansion. Hence, the identification and functional analyses of Pst effectors are of great significance in deciphering the Pst pathogenicity mechanism. Here, we identified one candidate Pst effector Pst_9302 that could suppress Bax-triggered cell death in Nicotiana benthamiana. qRT-PCR analyses showed that the transcript levels of Pst_9302 were highly increased during the early infection stages of Pst. The transient expression of Pst_9302 in wheat via the type-three secretion system (T3SS) significantly inhibited the callose deposition induced by Pseudomonas syringae EtHAn. During wheat–Pst interaction, Pst_9302 overexpression suppressed reactive oxygen species (ROS) accumulation and cell death caused by the avirulent Pst race CYR23. The host-induced gene silencing (HIGS) of Pst_9302 resulted in decreased Pst pathogenicity with reduced infection area. The results suggest that Pst_9302 plays a virulence role in suppressing plant immunity and promoting Pst pathogenicity. Moreover, wheat voltage-dependent anion channel 1 protein (TaVDAC1) was identified as candidate Pst_9302-interacting proteins by yeast two-hybrid (Y2H) screening. Pull-down assays using the His-Pst_9302 and GST-TaVDAC1 protein verified their interactions. These results suggest that Pst_9302 may modulate wheat TaVDAC1 to regulate plant immunity.


Introduction
In nature, plants face threats from the environment and microbes.To defend against pathogenic microbes, plants have evolved a two-layered immune system [1].Pattern recognition receptors (PRRs) located on plant plasma membrane perceive the conserved pathogen/damage/microbe-associated molecular patterns (PAMPs/DAMPs/MAMPs) to activate the first layer immunity, called PAMP-triggered immunity (PTI).Surface pattern recognition receptors mainly refer to receptor-like proteins or receptor-like kinases, such as FLS2 and RXEG1 [2,3].For survival, pathogenic microorganisms secrete effectors into host cells where they overcome plant immunity, resulting in effector-triggered susceptibility (ETS) [1].In turn, plants have further evolved resistance proteins (R), known as intracellular nucleotide-bound leucine-rich repeat receptors (NLRs), for the perception of specific avirulence (Avr) proteins via direct ligand-receptor interactions or the indirect detection of effector activity, leading to effector-triggered immunity (ETI) [4].ETI is characterized with strong and localized hypersensitive cell death (HR) and reactive oxygen species (ROS) Plants 2024, 13, 94 2 of 16 burst, which is particularly effective against biotrophic pathogens that are entirely dependent on living host cells.Plant immunity is a holistic system in which PTI and ETI are not independent but contribute to each other [5][6][7].
Uncovering molecular mechanisms underlying pathogen pathogenicity could lay the foundation for engineering new strategies for improving disease resistance in crop plants by preventing pathogens from regulating host plant immunity.Effectors as important pathogenicity factors have been the focus in the deciphering of pathogen pathogenicity.Effectors are expressed at different infection stages and functional in different host plant cellular components [8], which may influence various biological processes, facilitating pathogen proliferation and dispersal.Studies have shown that plant pathogen effector proteins can function to interfere with the host hormone metabolism pathway, ubiquitination pathway, gene transcription, RNA processing, HR, etc. [9][10][11][12][13][14].
Puccinia striiformis f. sp.Tritici (Pst), the causal agent of wheat stripe rust, devastates wheat crops worldwide [15,16].Pst is an obligate biotrophic pathogen that establishes an elaborate parasitic relationship with its host plants.During infection, Pst forms haustoria to penetrate host cell walls, contact host cell membranes, and enable nutrient uptake [17,18].Like many other plant pathogens, rust fungi secrete effector proteins into the host cells [19].In spite of the importance in understanding the rust-wheat pathosystem, the lack of a stable transformation system limits the study of wheat rust fungal effectors [20].An alternative approach for effector screening developed is using a Pseudomonas fluorescens (Pf ) strain EtHAn (effector-to-host-analyzer) to deliver oomycete or fungal effectors into host cells via the bacteria type-three secretion system (T3SS) [21].The developed hostinduced gene silencing (HIGS) technique also provides a powerful tool for knocking down the expression of Pst effectors to study their roles in Pst pathogenicity [22,23].With the sequencing of the genome and haustorium transcriptome of the stripe rust fungus, a batch of effectors have been identified and the functional mechanism of certain effectors have been characterized [24,25].Studies show that Pst effectors are able to translocate into different plant cell compartments to disturb plant immunity [12,[26][27][28][29]. Pst_12806 enters into wheat chloroplasts where it interacts with the cytochrome b6f complex element TaISP to affect chloroplast metabolism and ROS production, thereby promoting Pst infection [28].Interestingly, another two Pst effectors, Pst_4 and Pst_5, also interact with TaISP in the cytoplasm to prevent TaISP from entering chloroplasts [29].Pst_A23 enters the wheat splicesome to directly bind the splicing sites of wheat pre-mRNAs and reprogram wheat pre-mRNA alternative splicing [12].PstGTA1 can locate in the wheat cell nucleus, which binds to the promoter region of the wheat susceptibility gene TaSIG to activate its transcription by regulating the acetylation level of H3K4 [30].The unconventional effector PsSpg1 enhances the phosphorylation of the wheat susceptibility gene TaPsIPK1 to promote the nuclear entrance of TaPsIPK1, where it modulates the transcription activity of TaCBF1 and promotes wheat susceptibility [31].
In this study, from the genome of Pst race CYR32 [25], a candidate effector Pst_9302 that is highly induced at the early infection stage of Pst was identified.Pst_9302 specifically exists in Pst and is conserved among different Pst isolates.The transient expression of Pst_9302 inhibited Bax-induced cell death in Nicotiana benthamiana.The delivery of Pst_9302 into wheat cells via EtHAn suppressed the PTI-associated callose deposition caused by nonpathogenic bacteria.Upon avirulent Pst challenge, Pst_9302 overexpression inhibits wheat ETI, resulting in decreased ROS accumulation and HR occurrence.These data indicated the virulence function of Pst_9302 in suppressing host immunity, including both PTI and ETI.Furthermore, the host-induced gene silencing (HIGS) of Pst_9302 decreased the virulence of Pst, resulting in reduced uredium formation and attenuated fungal infection area.The yeast two-hybrid (Y2H) assay identified that the wheat voltage-dependent anion channel 1 protein TaVDAC1 was able to interact with Pst_9302, and the interactions between them were verified via the pull-down assay.VDAC is a major outer mitochondrial membrane protein, which is reported to participate in mitochondrial-mediated apoptosis.Overall, our Plants 2024, 13, 94 3 of 16 data suggest that Pst_9302 is an important Pst pathogenicity factor which likely regulates plant immunity by targeting the mitochondrial VDAC in plants.

Pst_9302 Is Induced during Pst Infection in Wheat
From the genome of the Pst race CYR32, Pst_9302 encoding a secreted protein was identified, with an open reading frame of 372 bp.SignalP 5.0 predicted that Pst_9302 contains a signal peptide encoded by the N-terminal 1-21 amino acids (Figure S1).The mature protein of Pst_9302 contains no transmembrane domain or conserved domain.BlastN analyses revealed no homologues genes in either Puccinia triticina or Puccinia graminis, the other two rust fungal pathogens in wheat, or in other pathogenic fungi.Among 13 tested Pst isolates from different countries, seven nucleotides in the ORF of Pst_9302 showed variations (Figure 1A), which resulted in variations in five amino acids (Figure 1B).The results showed that Pst_9302 is a Pst-specific secreted protein with low polymorphism within Pst.To determine whether Pst_9302 is involved in Pst infection, we measured its transcript levels at different Pst infection stages.qRT-PCR analyses showed that Pst_9302 was highly induced in planta infection of Pst, reaching the peak at 18 h post inoculation (hpi), and was 23-fold higher than that in urediniospores (Figure 2).At 18 hpi, Pst begins to form the initial haustorium, suggesting the role of Pst_9302 at the early infection stage of Pst.portant Pst pathogenicity factor which likely regulates plant immunity by targeting the mitochondrial VDAC in plants.

Pst_9302 Is Induced during Pst Infection in Wheat
From the genome of the Pst race CYR32, Pst_9302 encoding a secreted protein was identified, with an open reading frame of 372 bp.SignalP 5.0 predicted that Pst_9302 contains a signal peptide encoded by the N-terminal 1-21 amino acids (Figure S1).The mature protein of Pst_9302 contains no transmembrane domain or conserved domain.BlastN analyses revealed no homologues genes in either Puccinia triticina or Puccinia graminis, the other two rust fungal pathogens in wheat, or in other pathogenic fungi.Among 13 tested Pst isolates from different countries, seven nucleotides in the ORF of Pst_9302 showed variations (Figure 1A), which resulted in variations in five amino acids (Figure 1B).The results showed that Pst_9302 is a Pst-specific secreted protein with low polymorphism within Pst.To determine whether Pst_9302 is involved in Pst infection, we measured its transcript levels at different Pst infection stages.qRT-PCR analyses showed that Pst_9302 was highly induced in planta infection of Pst, reaching the peak at 18 h post inoculation (hpi), and was 23-fold higher than that in urediniospores (Figure 2).At 18 hpi, Pst begins to form the initial haustorium, suggesting the role of Pst_9302 at the early infection stage of Pst.

Pst_9302 Is a Candidate Effector That Is Able to Suppress Bax-Induced Cell Death in Planta
The mouse proapoptotic factor Bax is reported to induce programmed cell death (PCD) that is similar to HR in plants [14,32].Thereby, detecting the suppression ability of Bax-triggered cell death has become a high-throughput and effective assay to determine the virulence function of pathogen effectors in inhibiting plant defense responses [33,34].Here, obvious cell death was observed in N. benthamiana leaves expressing Bax mediated via Agrobacterium infiltration (Figure 3).In N. benthamiana leaves infiltrated with Agrobacterium carrying Pst_9302 without the signal peptide (Pst_9302 ΔSP ) 24 h before Bax infiltration, no cell death was observed (Figure 3).In contrast, in N. benthamiana leaves transiently expressing the negative control EV (empty vector), Bax-induced cell death was not affected (Figure 3).The results showed that the transient expression of Pst_9302 suppressed Bax-induced cell death.Pst_9302 suppresses Bax-triggered cell death in N. benthamiana.N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying various constructs, as labeled in the schematic drawing.For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ΔSP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Pst_9302 Inhibits PTI in Wheat
In order to elucidate the virulence function of effector Pst_9302 ΔSP in host wheat, Pst_9302 ΔSP was cloned into the effector detector vector (DEV) pEDV6 and delivered into wheat plants via a modified Pseudomonas fluorescens EtHAn strain.EtHAn and the

Pst_9302 Is a Candidate Effector That Is Able to Suppress Bax-Induced Cell Death in Planta
The mouse proapoptotic factor Bax is reported to induce programmed cell death (PCD) that is similar to HR in plants [14,32].Thereby, detecting the suppression ability of Bax-triggered cell death has become a high-throughput and effective assay to determine the virulence function of pathogen effectors in inhibiting plant defense responses [33,34].Here, obvious cell death was observed in N. benthamiana leaves expressing Bax mediated via Agrobacterium infiltration (Figure 3).In N. benthamiana leaves infiltrated with Agrobacterium carrying Pst_9302 without the signal peptide (Pst_9302 ∆SP ) 24 h before Bax infiltration, no cell death was observed (Figure 3).In contrast, in N. benthamiana leaves transiently expressing the negative control EV (empty vector), Bax-induced cell death was not affected (Figure 3).The results showed that the transient expression of Pst_9302 suppressed Baxinduced cell death.

Pst_9302 Is a Candidate Effector That Is Able to Suppress Bax-Induced Cell Death in Planta
The mouse proapoptotic factor Bax is reported to induce programmed cell death (PCD) that is similar to HR in plants [14,32].Thereby, detecting the suppression ability of Bax-triggered cell death has become a high-throughput and effective assay to determine the virulence function of pathogen effectors in inhibiting plant defense responses [33,34].Here, obvious cell death was observed in N. benthamiana leaves expressing Bax mediated via Agrobacterium infiltration (Figure 3).In N. benthamiana leaves infiltrated with Agrobacterium carrying Pst_9302 without the signal peptide (Pst_9302 ΔSP ) 24 h before Bax infiltration, no cell death was observed (Figure 3).In contrast, in N. benthamiana leaves transiently expressing the negative control EV (empty vector), Bax-induced cell death was not affected (Figure 3).The results showed that the transient expression of Pst_9302 suppressed Bax-induced cell death.For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ΔSP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Pst_9302 Inhibits PTI in Wheat
In order to elucidate the virulence function of effector Pst_9302 ΔSP in host wheat, Pst_9302 ΔSP was cloned into the effector detector vector (DEV) pEDV6 and delivered into wheat plants via a modified Pseudomonas fluorescens EtHAn strain.EtHAn and the For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ∆SP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Pst_9302 Inhibits PTI in Wheat
In order to elucidate the virulence function of effector Pst_9302 ∆SP in host wheat, Pst_9302 ∆SP was cloned into the effector detector vector (DEV) pEDV6 and delivered into wheat plants via a modified Pseudomonas fluorescens EtHAn strain.EtHAn and the transformants of EtHAn carrying DsRed were used as the negative controls.Necrotic or chlorotic reactions were not observed in any of the wheat leaves infiltrated with buffer, EtHAn, EtHAn carrying DsRed, or Pst_9302 ∆SP (Figure 4A).Necrosis was observed in leaves infiltrated with EtHAn carrying AvrRpt2, an avirulent gene of P. fluorescens, along with abundant callose deposition (Figure 4A,B).AvrRpt2, which is known to trigger cell death in wheat, served as a positive control.The observed necrotic and callose accumulation caused by AvrRpt2 indicate success of the system.Callose deposition was observed in wheat infiltrated with EtHAn (Figure 4B), indicating that infection with non-pathogenic EtHAn triggers PTI in wheat.In wheat leaves expressing dsRed, no obvious change in callose deposition was observed compared to in the control plants infiltrated with EtHAn (Figure 4B,C).In contrast, under the same conditions, leaves inoculated with EtHAn carrying Pst_9302 ∆SP exhibited less callose accumulation compared to those of the negative controls (Figure 4B,C).The results indicate that the Pst effector Pst_9302 could partially suppress PTI in wheat.

Pst_9302 Suppresses Wheat ETI
Because many pathogen effectors have developed the ability to suppress ETI during evolution, we delivered Pst_9302 ∆SP into the wheat cultivar Suwon11 to evaluate ETI resistance to the avirulent Pst race CYR23.The area of reactive oxygen species (ROS) burst and necrosis area per infection site were observed and counted.DAB staining revealed that Plants 2024, 13, 94 6 of 16 ROS accumulation per infection site in wheat leaves transiently expressing Pst_9302 ∆SP was reduced by 44.0% and 41.8% compared to that in wheat leaves expressing DsRed at 24 hpi and 48 hpi (Figure 5A,B).A hypersensitive response was observed under fluorescent microscopy and the fluorescence areas were calculated.Compared with the wheat leaves expressing DsRed, the necrotic cell area in wheat leaves expressing Pst_9302 ∆SP was decreased by 29.8% at 24 hpi (Figure 5A,C).Our results suggest that Pst_9302 ∆SP overexpression is able to suppress the ETI-triggered ROS accumulation of the avirulent Pst race in wheat.

Pst_9302 Suppresses Wheat ETI
Because many pathogen effectors have developed the ability to suppress ETI during evolution, we delivered Pst_9302 ΔSP into the wheat cultivar Suwon11 to evaluate ETI resistance to the avirulent Pst race CYR23.The area of reactive oxygen species (ROS) burst and necrosis area per infection site were observed and counted.DAB staining revealed that ROS accumulation per infection site in wheat leaves transiently expressing Pst_9302 ΔSP was reduced by 44.0% and 41.8% compared to that in wheat leaves expressing DsRed at 24 hpi and 48 hpi (Figure 5A,B).A hypersensitive response was observed under fluorescent microscopy and the fluorescence areas were calculated.Compared with the wheat leaves expressing DsRed, the necrotic cell area in wheat leaves expressing Pst_9302 ΔSP was decreased by 29.8% at 24 hpi (Figure 5A,C).Our results suggest that Pst_9302 ΔSP overexpression is able to suppress the ETI-triggered ROS accumulation of the avirulent Pst race in wheat.

Silencing of Pst_9302 Reduces Pst Pathogenicity
To further characterize the function of Pst_9302 in Pst pathogenicity, we knocked down the expression of Pst_9302 during wheat-Pst interaction using barley stripe mosaic virus (BSMV)-mediated host-induced gene silencing (HIGS).One specific silencing fragment of Pst_9302 was designed to generate the recombinant virus constructs.The BSMV: γ-TaPDS was used as the virus indicator.At 10 days post BSMV inoculation, BSMV: γ-TaPDSinoculated wheat leaves showed photobleaching, indicating the successful work of the virus system.Then, the fourth leaves of BSMV inoculated wheat plants were further challenged with Pst race CYR32, which was virulent on the control wheat plants.The disease Plants 2024, 13, 94 7 of 16 phenotypes were observed at 14 dpi.BSMV: γ-inoculated wheat leaves were susceptible to Pst race CYR32 with abundant uredium formed, and in BSMV: Pst_9302-inoculated wheat leaves, decreased uredium was formed (Figure 6A).qRT-PCR analyses showed that the expression level of Pst_9302 was reduced by 60-75% in Pst_9302 knockdown plants compared to in the control plants (Figure 6B).Histological observation revealed that the hyphal branch per infection site was significantly reduced in Pst_9302-silencing wheat plants at 48 hpi compared to that in the control leaves (Figure 7A-C).At both 48 hpi and 120 hpi, the Pst infection areas were significantly decreased in Pst_9302-silencing wheat plants compared to those in the control leaves (Figure 7D,E).The data showed that knocking down the expression of Pst_9302 weakened Pst pathogenicity, indicating the contribution of Pst_9302 in stripe rust fungal virulence.
cessful work of the virus system.Then, the fourth leaves of BSMV inoc plants were further challenged with Pst race CYR32, which was virulent o wheat plants.The disease phenotypes were observed at 14 dpi.BSMV: wheat leaves were susceptible to Pst race CYR32 with abundant uredium fo BSMV: Pst_9302-inoculated wheat leaves, decreased uredium was formed qRT-PCR analyses showed that the expression level of Pst_9302 was reduce in Pst_9302 knockdown plants compared to in the control plants (Figure 6B) observation revealed that the hyphal branch per infection site was significa in Pst_9302-silencing wheat plants at 48 hpi compared to that in the control l 7A-C).At both 48 hpi and 120 hpi, the Pst infection areas were significantly Pst_9302-silencing wheat plants compared to those in the control leaves ( The data showed that knocking down the expression of Pst_9302 weaken genicity, indicating the contribution of Pst_9302 in stripe rust fungal virulen

Pst_9302 Interacts with TaVDAC1 In Vitro and In Vivo
To understand the virulence function mechanism of Pst_9302 in suppressing wheat immunity, a yeast two-hybrid (Y2H) assay was performed to identify the potential host targets of Pst_9302.With Pst_9302 ΔSP as the bait, 18 distinct putative wheat targets with known annotation were identified from a wheat-Pst cDNA library.The coding sequence of six interacting candidate genes was successfully cloned into pGADT7.The recombinant pGADT7 constructs were co-transformed with pGBKT7-Pst_9302 ΔSP to detect their interaction via Y2H.Yeast cells expressing Pst_9302 ΔSP and wheat protein encoding voltage-dependent anion channels, named TaVDAC1, grew and turned blue on the selection medium, SD/-Trp/-Leu/-His/-Ade with X-a-gal added (Figure 8A), indicating the interaction between Pst_9302 and TaVDAC1.To determine whether Pst_9302 can directly interact with TaVDAC1, a pull-down assay was conducted.GST-TaVDAC1 or GST protein was co-incubated with Pst_9302-His and precipitated using His beads.The immunoprecipitated protein complexes were detected via Western blotting using anti-His

Pst_9302 Interacts with TaVDAC1 In Vitro and In Vivo
To understand the virulence function mechanism of Pst_9302 in suppressing wheat immunity, a yeast two-hybrid (Y2H) assay was performed to identify the potential host targets of Pst_9302.With Pst_9302 ∆SP as the bait, 18 distinct putative wheat targets with known annotation were identified from a wheat-Pst cDNA library.The coding sequence of six interacting candidate genes was successfully cloned into pGADT7.The recombinant pGADT7 constructs were co-transformed with pGBKT7-Pst_9302 ∆SP to detect their interaction via Y2H.Yeast cells expressing Pst_9302 ∆SP and wheat protein encoding voltagedependent anion channels, named TaVDAC1, grew and turned blue on the selection medium, SD/-Trp/-Leu/-His/-Ade with X-a-gal added (Figure 8A), indicating the interaction between Pst_9302 and TaVDAC1.To determine whether Pst_9302 can directly interact with TaVDAC1, a pull-down assay was conducted.GST-TaVDAC1 or GST protein was co-incubated with Pst_9302-His and precipitated using His beads.The immunoprecipitated protein complexes were detected via Western blotting using anti-His antibody and anti-GST antibody.The results showed that GST-TaVDAC1 was able to interact with His-Pst_9302, whereas GST alone could not (Figure 8B).antibody and anti-GST antibody.The results showed that GST-TaVDAC1 was able to interact with His-Pst_9302, whereas GST alone could not (Figure 8B).

Discussion
During infection, wheat stripe rust fungus secrets clusters of effectors via haustorium to host cells to regulate plant immunity and metabolism, thus promoting host plant susceptibility.Identifying the key effectors of Pst is of great significance in revealing the pathogenic mechanism of rust fungi.The completed Pst genome and haustorial transcriptome identified a large quantity of secreted proteins [24,25].Developed functional analyses systems, including EtHAn-mediated rust fungal effector expression in wheat and HIGS, lay the foundation for analyzing the function of rust fungal effectors [35,36].Via HIGS-and T3SS-mediated effector delivery technology, a series of Pst effectors that significantly affect Pst virulence have been identified [26][27][28][29]37,38].In this study, one Pst effector, Pst_9302, which is highly induced at 18 hpi during Pst infection in wheat, was identified.During Pst infection, after landing on the wheat surface, the urediniospores germinate and penetrate through the stoma to form sub-stomatal vesicles, then the initial infection hyphae were formed at 12 hpi and began to form an initial haustorium at 18 hpi.Therefore, the high transcript level of Pst_9302 at 18 hpi indicates the possible role of Pst_9302 during the early establishment of the parasitic relationship of Pst in host plants.BlastN analyses showed that Pst_9302 is a Pst-specific effector, which is conservative among Pst isolates, indicating that Pst_9302 may function as a common virulent factor in Pst but may be lacking in other rust fungi.

Discussion
During infection, wheat stripe rust fungus secrets clusters of effectors via haustorium to host cells to regulate plant immunity and metabolism, thus promoting host plant susceptibility.Identifying the key effectors of Pst is of great significance in revealing the pathogenic mechanism of rust fungi.The completed Pst genome and haustorial transcriptome identified a large quantity of secreted proteins [24,25].Developed functional analyses systems, including EtHAn-mediated rust fungal effector expression in wheat and HIGS, lay the foundation for analyzing the function of rust fungal effectors [35,36].Via HIGS-and T3SS-mediated effector delivery technology, a series of Pst effectors that significantly affect Pst virulence have been identified [26][27][28][29]37,38].In this study, one Pst effector, Pst_9302, which is highly induced at 18 hpi during Pst infection in wheat, was identified.During Pst infection, after landing on the wheat surface, the urediniospores germinate and penetrate through the stoma to form sub-stomatal vesicles, then the initial infection hyphae were formed at 12 hpi and began to form an initial haustorium at 18 hpi.Therefore, the high transcript level of Pst_9302 at 18 hpi indicates the possible role of Pst_9302 during the early establishment of the parasitic relationship of Pst in host plants.BlastN analyses showed that Pst_9302 is a Pst-specific effector, which is conservative among Pst isolates, indicating that Pst_9302 may function as a common virulent factor in Pst but may be lacking in other rust fungi.
The ability to suppress Bax-triggered PCD is a valuable initial screen for pathogen effectors because it physiologically resembles defense-related HR [32].Although it is possible that the use of a non-host species could result in some responses not relevant to Pst infection, the ability to suppress PCD triggered by Bax indicates that Pst_9302 is able to suppress PCD in plants.Pst_9302 is able to suppress callose deposition elicited by the non-pathogenic Pf strain EtHAn.Callose deposition is a hallmark of PTI.The suppression of callose deposition by Pst_9302 indicates that it can inhibit host PTI.In fact, as the first line of defense, PTI involves multiple processes that can be attenuated by diverse pathogens to achieve further infection.Most effectors of the oomycete Hyaloperonospora arabidopsidis and bacterial Psedomonas syring pv.tomato DC3000 (PtoDC3000) can suppress PTI in plants elicited by non-pathogenic microorganisms and PAMPs in different ways [39][40][41].For example, the Hyaloperonospora arabidopsidis effector HaRxL96 and its homologue PsAvh163 in Phytophthora sojaeare could both suppress immunity in soybean.HaRxL96 inhibits immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in N. benthamiana that is dependent on RAR1 and Hsp90.1 [39].Oomycete effector AVRblb2 interacts with cyclic nucleotide-gated channels using calcium sensors as cofactors to inhibit calcium channel activation and suppress pattern-triggered immunity [41].Bacterial effectors are also reported to suppress plant immunity by targeting PTI signaling components.Ralstonia solanacearum subverts plant PTI using multiple effectors, among which RipE1 manipulates JA signaling and SA synthesis to promote infection [42].The Pst effector HASP98 targets and inhibits the kinase activity of wheat TaMAPK6, the key component of the plant basal defense pathway, to suppress wheat immunity [26].It is possible that various Pst effectors function by targeting different conserved components of the PTI signaling pathway.
Pst_9302 also significantly suppress ETI responses in wheat plants challenged with the avirulent Pst isolate CYR23.Because biotrophs must actively suppress PCD in host plants to propagate, the suppression of ETI responses observed in this study demonstrates their virulence functions.Recent studies reported that plant PTI and ETI are not distinctive pathways but are able to potentiate each other.The inactivation of the key receptors in PTI also abolished ETI [6].Thus, the suppression ETI by Pst_9302 may partially result from the inhibited PTI.
HIGS is a useful tool to study the function of effectors in biotrophic fungi, which enable the identification of effectors that play a majority function in pathogen pathogenicity.For example, the HIGSs of Pst_13661, Pst_A23, Pst_4, Pst_5, etc., weaken Pst pathogenicity and increase wheat resistance to Pst [12,29,43].In our study, HIGS effectively silenced Pst_9302, and silenced plants produced fewer urediniospores.Infection area and hyphae branches were reduced.These results suggest that Pst_9302 functions as an important pathogenicity factor that contributes to Pst virulence.
The effector proteins of wheat stripe rust can regulate host immune response through various action pathways.Pst_12806, Pst_4, and Pst_5 inhibit chloroplast-mediated immunity by interfering with photosynthesis and promoting pathogenicity [28,29].The Pst effector PstGSRE1 is rich in glycine and serine and regulates host gene transcription by stopping transcription factor TaLOL2 from entering into the nucleus [27].The nuclear localized effectors Pst_A23 and PstGTA regulate host gene expression via post-transcription modification, by modulating host pre-mRNA alternative splicing or the H3K4 acetylation level of TaSIG to promote fungal infection [12,30].Pst18363 and Pst27791 enhance the activity of the negative immune regulatory factor Nudix hydrolase23 (TaNUDX23) and the stability of TaRaf46, while PsSpg1 inhibits host disease resistance by increasing the kinase activity of the susceptible gene TaPsIPK1 [31,44,45].These effectors manipulate susceptible host genes to mediate wheat susceptibility, while another type of effector protein suppresses host immunity by inhibiting the activity of immune positive regulatory factors.Hasp98 and PstGSRE4 inhibit the activity of the host immune positive regulatory kinase TaMAPK4 and the copper zinc superoxide dismutase TaCZSOD2, respectively, leading to a decrease in the accumulation of reactive oxygen species in the host [26,46].Voltage-dependent anion channels (VDACs), a component of the outer membrane of mitochondria, were first identified in yeast and are present in all organisms [47].VDACs regulate the transport of substances between cytoplasm and mitochondria, e.g., K + , Cl − , Ca 2+ , HPO 4 2− , ATP 4− , O 2− , and mitochondria-mediated cell death [46].In plants, studies have shown that VDACs not only affect plant development but also positively regulate pathogen defense [48].Arabidopsis AtVDAC1 regulates infection resistance to Agrobacterium tumefaciens.NbVDACs are involved in resistance to Pseudomonas cichorii [49].Notably, VDACs also function in Bax-mediated cell death [50,51].Given these results, we hypothesize that Pst_9302 suppresses Bax-induced cell death and plant immunity via association with plant mitochondrial VDACs.Further studies are necessary to investigate the role of TaVDAC1 in plant cell death and defense response to Pst infection and analyze the regulation mechanism between Pst_9302 and TaVDAC1.

Plant Materials, Fungal Isolates, and Bacterial Strains
Seedlings of wheat (Triticum aestivum L.) cultivar Suwon 11 (Su11) and N. benthamiana were grown in a greenhouse under 8/16 h night/day conditions at 16 • C and 22 • C, respectively.Pst races CYR23 and CYR32 were propagated on wheat cultivar Mingxian 169 and Suwon 11. A. tumefaciens strain GV3101 was cultured in LB medium at 28 • C for transient expression in N. benthamiana.P. fluorescens strain EtHAn was cultured in King's B medium at 28 • C and used to deliver effectors to wheat.Yeast strains AH109 were cultured in YPDA medium at 30 • C for Y2H assays.E. coli BL21(DE3) was used for protein expression.

RNA Isolation and qRT-PCR
To measure the expression levels of Pst_9302 in the Pst-infected wheat leaves, the leaf tissues were sampled at 18 h, 24 h, 48 h, 72 h, 120 h, 168 h, and 216 h and stored at −80 • C for RNA extraction.Frozen urediniospores, germinated tubes, and infected leaves were ground in liquid nitrogen, and RNA was isolated with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.RNA was reverse transcribed using a RevertAid First Stand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).RT-qPCR analysis was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).The elongation factor genes PstEF1 from Pst was used as internal references.Primers used in qRT-PCR are listed in Table S1.Relative expression levels of target genes were calculated using the comparative 2 −∆∆CT method [52].Each experiment was performed for three biological replications.

Plasmid Construction
For the transient expression of Pst_9302 in N. benthamiana, the coding sequence (CDS) of Pst_9302 without a signal peptide and the Bax gene were PCR amplified and inserted into the ClaI/NotI restriction sites in vector potato virus X (PVX) pGR106 to obtain the recombinant plasmids PVX-Pst_9302 ∆SP and PVX-Bax.
For transient expression in wheat via Pf strain EtHAn, sequences encoding mature proteins of Pst_9302 without the signal peptide were amplified from Pst race CYR32 cDNA and cloned into the pDONR221 vector (Invitrogen, Carlsbad, CA, USA).pDONR221 constructs were transferred to pEDV6 via a gateway LR recombination reaction (Invitrogen, Carlsbad, CA, USA).
For BSMV-mediated HIGSs of Pst_9302, a 142 bp fragment specific to Pst_9302 was designed using siRNA-Finder21 (Si-Fi21) software analysis and ligated to Not I/Pac I-digested BSMV: γ vector to construct the RNA-based derivative plasmids BSMV-Pst_9302-as.
In order to screen target proteins, sequence-encoding Pst_9302 mature proteins without the signal peptide were inserted into pGBKT7 to form BK-Pst_9302.The full-length cDNA sequence of the TaVDAC1 protein was inserted into pGADT7 to form AD-TaVDAC1.
To confirm the interaction between Pst_9302 and TaVDAC1, the pull-down assay was performed.The sequence-encoding-Pst_9302 ∆SP protein was cloned into pET32a, and the TaVDAC1 protein was cloned into pGEX4T-1.The primers used for all constructs are listed in Table S1.

A. tumefaciens Infiltration Assays for Suppression of Bax-Induced PCD
Constructs were introduced into the A. tumefaciens strain GV3101 via electroporation, and positive transformants were selected using kanamycin and rifampicin.Individual clones were verified via PCR.For infiltration into leaves, recombinant strains of A. tumefaciens were grown in LB medium for 48 h, harvested, washed three times with 10 mM MgCl 2 , resuspended in 10 mM MgCl 2 to a final OD600 = 0.2, and incubated at room temperature for 2 h prior to infiltration.Cell suspensions were infiltrated into N. benthamiana leaves using a syringe without needle.To assay the suppression of Bax-induced cell death, an A. tumefaciens cell suspension carrying Pst_9302 ∆SP was initially infiltrated.A. tumefaciens cells carrying Bax were infiltrated into the same site 24 h later.Symptoms were scored 3-7 days post infiltration (dpi) with Bax.Each assay consisted of at least three plants and three leaves and was performed in triplicate.

Bacterial TTSS-Mediated Delivery of Pst_9302 in Wheat Plants
Constructs were transformed into Pf EtHAn via electroporation.For transient expression in wheat cells, bacteria were grown overnight, collected, washed twice with sterile 10 mM MgCl 2 , and resuspended to an OD600 = 1.0.For the analysis of PTI suppression, the suspended cells were infiltrated into the second leaf of wheat cultivar Swon11.Leaves infiltrated with EtHAn carrying DsRed were used as negative control.At 24 h post inoculation, the infiltrated wheat leaves were collected and stained with aniline blue to count callose accumulation, which was measured as an indicator of PTI.
In ETI-suppression assays, the second leaves of wheat cultivar Suwon11 were initially infiltrated with recombinant EtHAn at an OD600 = 1.0.Twenty-four hours post infiltration (hpi), infected leaves were challenged with the avirulent Pst race CYR23 at the initial infiltration site.Challenged leaves were sampled at 24 and 48 hpi for histochemical observation.Oxidative burst and cell death occurrence were scored to evaluate the resistance response.Leaves infiltrated with EtHAn carrying DsRed were used as negative controls and all data were compared to results for the negative controls.

BSMV Mediate Host-Induced Gene Silencing
BSMV constructs (BSMV: Pst_9302 for the silencing of Pst_9302, BSMV: γ and BSMV: TaPDS) were used to inoculate the second leaves of the two-leaf stage wheat cultivar Swon11 [53].BSMV: γ was used as the negative control, and BSMV: TaPDS was used as a virus-positive control.Ten days after virus inoculation, the fourth leaves were further inoculated with the fresh urediniospores of Pst CYR32.The inoculated leaves were collected at 48, and 120 hpi for silencing efficiency analyses and the histological observation of fungal growth.Fourteen days after Pst inoculation, the phenotypes of the fourth leaves were examined and photographed.

Histochemical Analysis
To visualize callose deposition, wheat leaves were harvested, cleared, and stained with aniline blue [54].Briefly, the collected wheat leaves were immersed in ethyl alcohol:acetic acid (1:1 v/v) and chloral hydrate until the leaf segment was transparent, and were then stained with 0.05% aniline blue in 0.067 M K 2 HPO 4 (pH 9.6).Stained samples were observed with an Olympus BX-52 microscope (Olympus Corporation, Tokyo, Japan) under blue light excitation (excitation wavelength 450-480 nm, emission wavelength 515 nm).The number of callose depositions was calculated for 1 mm 2 in 50 replicates using ImageJ software.Hydrogen peroxide accumulation was monitored using (3,3 ′ -diaminobenzidine) DAB staining as previously described [55].The sampled Pst-infected leaves were incubated with 1.5 mg/mL DAB under illumination for 5 h at room temperature.H 2 O 2 accumulation was examined and recorded with an Olympus BX-52 microscope equipped with cellSens Entry software.The autofluorescence of attacked mesophyll cells was observed under a fluorescence microscope (excitation filter 485 nm, dichromic mirror 510 nm, barrier filter 520 nm) and measured using cellSens Entry software (Olympus, Tokyo, Japan).Wheat germ agglutinin (WGA) conjugated to Alexa 488 (Invitrogen) was used to stain the infection structure of Pst as previously described [56].The hyphal branch was measured as the number of observed infection hyphae formed from one substomatal vesicle (SV).The haustoria was measured as the number of observed haustoria formed at the tip of all infection hyphae from one SV.The infection area was calculated as the area of infection hyphae using CellSens Entry software (Olympus, Tokyo, Japan).Using cellSens Entry software (Olympus, Tokyo, Japan), the number of haustoria, hyphae branches, and hyphal infection areas were measured from 50 infection sites in which substomatal vesicles had formed underneath the stoma.

Yeast Two-Hybrid Assay
The recombinant Pst_9302 ∆SP -pGBKT7 vector was used as a bait to screen a Y2H cDNA library produced from Pst-infected wheat leaves.Yeast strain AH109 co-transformed with BK-Pst_9302 ∆SP and the library plasmids were cultured on SD/-Trp-Leu and screened on SD/-Trp-Leu-His-Ade and X-a-gal-supplemented medium.Positive colonies on SD/-Trp-Leu-His-Ade plates were used as templates for PCR, and the products were sequenced.The coding sequences of the candidate interacting proteins were cloned into pGADT7 and co-transformed with BK-Pst_9302 ∆SP into yeast strain AH109 to examine their interactions through growth on the SD/-Trp-Leu-His-Ade and X-a-gal-supplemented medium.

Protein Expression in E. coli and Purification
The recombinant plasmids PET32a-Pst_9302 ∆SP and PGEX4T-1-TaVDAC1 were transformed into E. coli BL21 (DE3).A single colony that had been freshly plated was inoculated into 40 mL of liquid LB media before undergoing shaking at 200 rpm at a temperature of 37 • C Then, the 40 mL culture was inoculated into 1 L of LB and cultured at 37 • C to OD 600 = 0.6.Then, 0.5 mM IPTG was added to the culture before undergoing additional shaking at 160 rpm at a temperature of 18 • C for 20 h.E. coli cells were harvested and resuspended in 50 mM Tris (pH 7.5), 150 mM NaCl, 20 mM β-mercaptoethanol (β-ME), and 1 mM PMSF and lysed via sonication.The lysates were centrifuged for 20 min at 20,000× g.The His-Pst_9302 ∆SP and GST-TaVDAC1 were purified via affinity chromatography using a HisTrap (Ni-NTA) column (GE Healthcare, Chicago, IL, USA) and GST-S Glutathione Sepharose 4B chromatography (GE Healthcare).

Pull-Down Assay
The interactions between His-Pst_9302 ∆SP and GST-TaVDAC1 were assessed via pulldown assays using a HisTrap (Ni-NTA) column (GE) according to the manufacturer's instructions.The input proteins and proteins that had been immunoprecipitated via Ni-NTA beads (His pull-down) were analyzed through immunoblotting with anti-GST-

Figure 1 .
Figure 1.Polymorphism analyses of Pst_9302 in 13 Pst isolates.(A).Nucleotide acid sequence alignment of Pst_9302 in 13 Pst isolates.The nucleotide acid sequences were downloaded from the NCBI database.(B).The amino acid sequence alignment of Pst_9302 from 13 Pst isolates.The blue code represents 100% similarity and cyan code represents 50% similarity.The numbers on the left represent the name of the Pst isolate.

Figure 1 .
Figure 1.Polymorphism analyses of Pst_9302 in 13 Pst isolates.(A).Nucleotide acid sequence alignment of Pst_9302 in 13 Pst isolates.The nucleotide acid sequences were downloaded from the NCBI database.(B).The amino acid sequence alignment of Pst_9302 from 13 Pst isolates.The blue code represents 100% similarity and cyan code represents 50% similarity.The numbers on the left represent the name of the Pst isolate.

Figure 2 .
Figure 2. Transcript profiles of Pst_9302 during Pst infection.The second leaves of wheat cultivar Swon11 were inoculated with virulent Pst race CYR32 and the samples were collected at 18, 24, 48, 72, 120, 168, and 216 h post inoculation (hpi).Urediniospores, U; germ tubes, GT.Pst elongation factor gene PsEF1 was used as the internal control gene.Data are mean ± SD from three biological replicates.Asterisks indicates significant difference using unpaired two-tailed Student's t-test via comparison to that in urediniospores (*, p < 0.05; **, p< 0.01).

Figure 3 .
Figure 3. Pst_9302 suppresses Bax-triggered cell death in N. benthamiana.N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying various constructs, as labeled in the schematic drawing.For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ΔSP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Figure 2 .
Figure 2. Transcript profiles of Pst_9302 during Pst infection.The second leaves of wheat cultivar Swon11 were inoculated with virulent Pst race CYR32 and the samples were collected at 18, 24, 48, 72, 120, 168, and 216 h post inoculation (hpi).Urediniospores, U; germ tubes, GT.Pst elongation factor gene PsEF1 was used as the internal control gene.Data are mean ± SD from three biological replicates.Asterisks indicates significant difference using unpaired two-tailed Student's t-test via comparison to that in urediniospores (*, p < 0.05; **, p < 0.01).

Plants 2024 , 17 Figure 2 .
Figure 2. Transcript profiles of Pst_9302 during Pst infection.The second leaves of wheat cultivar Swon11 were inoculated with virulent Pst race CYR32 and the samples were collected at 18, 24, 48, 72, 120, 168, and 216 h post inoculation (hpi).Urediniospores, U; germ tubes, GT.Pst elongation factor gene PsEF1 was used as the internal control gene.Data are mean ± SD from three biological replicates.Asterisks indicates significant difference using unpaired two-tailed Student's t-test via comparison to that in urediniospores (*, p < 0.05; **, p< 0.01).

Figure 3 .
Figure 3. Pst_9302 suppresses Bax-triggered cell death in N. benthamiana.N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying various constructs, as labeled in the schematic drawing.For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ΔSP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Figure 3 .
Figure 3. Pst_9302 suppresses Bax-triggered cell death in N. benthamiana.N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying various constructs, as labeled in the schematic drawing.For co-filtration assays, leaves were first infiltrated with Agrobacteria expressing Pst_9302 ∆SP without the signal peptide followed by infiltration with Agrobacteria expressing mouse Bax after 24 h.Photos were taken 5 days after infiltration.The empty vector (EV) pGR106 was used as negative control.The same leaf was decolorized with destained solution (right).

Plants 2024 ,
13,  x FOR PEER REVIEW 5 of 17 transformants of EtHAn carrying DsRed were used as the negative controls.Necrotic or chlorotic reactions were not observed in any of the wheat leaves infiltrated with buffer, EtHAn, EtHAn carrying DsRed, or Pst_9302 ΔSP (Figure4A).Necrosis was observed in leaves infiltrated with EtHAn carrying AvrRpt2, an avirulent gene of P. fluorescens, along with abundant callose deposition (Figure4A,B).AvrRpt2, which is known to trigger cell death in wheat, served as a positive control.The observed necrotic and callose accumulation caused by AvrRpt2 indicate success of the system.Callose deposition was observed in wheat infiltrated with EtHAn (Figure4B), indicating that infection with non-pathogenic EtHAn triggers PTI in wheat.In wheat leaves expressing dsRed, no obvious change in callose deposition was observed compared to in the control plants infiltrated with EtHAn (Figure4B,C).In contrast, under the same conditions, leaves inoculated with EtHAn carrying Pst_9302 ΔSP exhibited less callose accumulation compared to those of the negative controls (Figure4B,C).The results indicate that the Pst effector Pst_9302 could partially suppress PTI in wheat.

Figure 4 .
Figure 4. Overexpression of Pst_9302 ΔSP suppresses PTI-associated callose deposition in wheat leaves challenged by non-pathogenic bacteria.(A) Representative wheat leaves inoculated with MgCl2 buffer, EtHAn, or EtHAn carrying pEDV6: DsRed, pEDV6: Pst_9302 ΔSP , and positive control pEDV6: AvrRpt2.Photos were taken at 48 h after bacterial infiltration.(B) Wheat leaves in (A) were sampled at 48 h and examined for callose deposition via epifluorescence microscopy after aniline blue staining.Bars = 10 μm.(C) Average numbers of callose deposits/mm 2 in wheat leaves infiltrated with bacteria expressing the indicated genes were calculated using ImageJ 2 software.MgCl2, EtHAn, and EtHAn carrying DsRed were used as controls.Data are mean ± SD from three biological replicates.Significance was analyzed using Duncan's new multiple range tests.Different lowercase letters indicate differences with a significance level of 0.05.

Figure 4 .
Figure 4. Overexpression of Pst_9302 ∆SP suppresses PTI-associated callose deposition in wheat leaves challenged by non-pathogenic bacteria.(A) Representative wheat leaves inoculated with MgCl 2 buffer, EtHAn, or EtHAn carrying pEDV6: DsRed, pEDV6: Pst_9302 ∆SP , and positive control pEDV6: AvrRpt2.Photos were taken at 48 h after bacterial infiltration.(B) Wheat leaves in (A) were sampled at 48 h and examined for callose deposition via epifluorescence microscopy after aniline blue staining.Bars = 10 µm.(C) Average numbers of callose deposits/mm 2 in wheat leaves infiltrated with bacteria expressing the indicated genes were calculated using ImageJ 2 software.MgCl 2 , EtHAn, and EtHAn carrying DsRed were used as controls.Data are mean ± SD from three biological replicates.Significance was analyzed using Duncan's new multiple range tests.Different lowercase letters indicate differences with a significance level of 0.05.

Figure 5 .
Figure 5. Overexpression of Pst_9302 ΔSP in wheat cells suppressed reactive oxygen species (ROS) accumulation and hypersensitive response triggered by the avirulent Pst CYR23.(A) The second leaves of wheat seedlings of cultivar Suwon11 were first infiltrated with EtHAn carrying Pst_9302 ΔSP at OD600 = 1.0 and then challenged with avirulent Pst race CYR23 24 h later.Inoculated leaves were sampled at 24 and 48 hpi and stained with 3,3′-diaminobenzidine for ROS detection and necrotic area at the infection sites, then observed under an Olympus BX-52 microscope.Black arrows indicate the substomatal vesicle of Pst, red arrows indicate the ROS and cell death area.Area of ROS (B) and necrotic cell death (C) per infection site were measured with cellSens_V4.1.1Entry software (Olympus, Tokyo, Japan).Data are mean ± SD (n = 50).Significance was analyzed using an unpaired two-tailed Student's t-test (**, p < 0.01).

Figure 5 .
Figure 5. Overexpression of Pst_9302 ∆SP in wheat cells suppressed reactive oxygen species (ROS) accumulation and hypersensitive response triggered by the avirulent Pst CYR23.(A) The second leaves of wheat seedlings of cultivar Suwon11 were first infiltrated with EtHAn carrying Pst_9302 ∆SP at OD600 = 1.0 and then challenged with avirulent Pst race CYR23 24 h later.Inoculated leaves were sampled at 24 and 48 hpi and stained with 3,3 ′ -diaminobenzidine for ROS detection and necrotic area at the infection sites, then observed under an Olympus BX-52 microscope.Black arrows indicate the substomatal vesicle of Pst, red arrows indicate the ROS and cell death area.Area of ROS (B) and necrotic cell death (C) per infection site were measured with cellSens_V4.1.1Entry software (Olympus, Tokyo, Japan).Data are mean ± SD (n = 50).Significance was analyzed using an unpaired two-tailed Student's t-test (**, p < 0.01).

Figure 6 .
Figure 6.BSMV-mediated HIGS of Pst_9302 weakened Pst pathogenicity.(A) Dise of Pst_9302 knockdown plants inoculated with virulent Pst race CYR32.The fourth l plants inoculated with BSMV: γ and BSMV: Pst_9302 were inoculated with Pst rac days post inoculation of BSMV.Disease phenotypes were observed at 14 days post Pst.(B) Silencing efficiency of Pst_9302 at 48 and 120 hpi analyzed via qRT-PCR.D with SD from three biological replicates.**, p< 0.01 via an unpaired two-tailed Stude

Figure 6 .
Figure 6.BSMV-mediated HIGS of Pst_9302 weakened Pst pathogenicity.(A) Disease symptoms of Pst_9302 knockdown plants inoculated with virulent Pst race CYR32.The fourth leaves of wheat plants inoculated with BSMV: γ and BSMV: Pst_9302 were inoculated with Pst race CYR32 at 10 days post inoculation of BSMV.Disease phenotypes were observed at 14 days post inoculation of Pst.(B) Silencing efficiency of Pst_9302 at 48 and 120 hpi analyzed via qRT-PCR.Data are means with SD from three biological replicates.**, p < 0.01 via an unpaired two-tailed Student's t-test.

Figure 7 .
Figure 7. Knockdown of Pst_9302 attenuated Pst growth and development.The Pst structures in Pst_9302 knockdown plants and the control plants were stained with wheat germ agglutinin conjugated to Alexa 488 (WGA) at 48 hpi and 120 hpi and observed under a fluorescence microscope.(A) Histological observation of Pst growth in Pst_9302-silenced wheat plants at 48 hpi.SV, sub-stomatal vesicle; IH, infection hyphae; HMC, haustorial mother cell; H, haustorium.Bars = 20 μm.(B,C) The hyphal branch and haustorium numbers of Pst per infection site at 48 hpi were measured using CellSens Entry software (Olympus, Tokyo, Japan) (D,E) Infection area of Pst in Pst_9302-silenced wheat plants at 48 and 120 hpi measured using CellSens Entry software (Olympus, Tokyo, Japan).Data are means ± SD (n = 50).*, p < 0.05; **, p < 0.01 using an unpaired two-tailed Student's t-test.

Figure 7 .
Figure 7. Knockdown of Pst_9302 attenuated Pst growth and development.The Pst structures in Pst_9302 knockdown plants and the control plants were stained with wheat germ agglutinin conjugated to Alexa 488 (WGA) at 48 hpi and 120 hpi and observed under a fluorescence microscope.(A) Histological observation of Pst growth in Pst_9302-silenced wheat plants at 48 hpi.SV, substomatal vesicle; IH, infection hyphae; HMC, haustorial mother cell; H, haustorium.Bars = 20 µm.(B,C) The hyphal branch and haustorium numbers of Pst per infection site at 48 hpi were measured using CellSens Entry software (Olympus, Tokyo, Japan) (D,E) Infection area of Pst in Pst_9302-silenced wheat plants at 48 and 120 hpi measured using CellSens Entry software (Olympus, Tokyo, Japan).Data are means ± SD (n = 50).*, p < 0.05; **, p < 0.01 using an unpaired two-tailed Student's t-test.

Figure 8 .
Figure 8. Pst_9302 interacts with TaVDAC1 in vitro and in vivo.(A) Interaction of Pst_9302 and TaVDAC1 by Y2H.A series of dilutions of yeast cells co-transforming BK-Pst_9302 ΔSP and AD-TaVDAC1 were incubated on SD-LW medium and SD-LWHA medium with X-α-gal.BK-Pst_9302 ΔSP and AD were used as the negative controls.SV40 large T-antigen (T) and murine p50 were used as the positive controls.(B) Interaction between Pst_9302-His and GST-TaVDAC1 via pull-down assay.The inputs and the immunoprecipitated protein complexes were detected via Western blot using anti-His antibody and anti-GST antibody.The red asterisks indicate the target protein.

Figure 8 .
Figure 8. Pst_9302 interacts with TaVDAC1 in vitro and in vivo.(A) Interaction of Pst_9302 and TaVDAC1 by Y2H.A series of dilutions of yeast cells co-transforming BK-Pst_9302 ∆SP and AD-TaVDAC1 were incubated on SD-LW medium and SD-LWHA medium with X-α-gal.BK-Pst_9302 ∆SP and AD were used as the negative controls.SV40 large T-antigen (T) and murine p50 were used as the positive controls.(B) Interaction between Pst_9302-His and GST-TaVDAC1 via pull-down assay.The inputs and the immunoprecipitated protein complexes were detected via Western blot using anti-His antibody and anti-GST antibody.The red asterisks indicate the target protein.