Molecular Plant Pathology

The compound appressoria of Sclerotinia sclerotiorum can produce cell wall‐degrading enzymes, effectors and toxins, which promote penetration and the death of host cells. Subsequently, invasive hyphae (IH) branch rapidly as necrotrophic growth and disease symptoms are observed. S. sclerotiorum can respond to complex stresses and regulate its metabolism to adapt to the external environment. Here we demonstrated that type 2C Ser/Thr phosphatase (PP2C) SsPtc3 responds to nutritional, osmotic, cell wall and oxidative stresses. Loss of function ΔSsptc3 mutants displayed defects in mycelial growth, sclerotia formation and reduced virulence. Phosphoproteomic analyses revealed that SsPtc3 is involved in autophagy and MAPK signalling pathways. We obtained evidence that SsPtc3 negatively modulates the phosphorylation of SsSmk1. SsSmk1 is essential for mycelial growth, compound appressorium formation and pathogenicity, SsPtc3 modulated phosphorylation homeostasis of SsSmk1 to maintain hyphal growth. SsPtc3 interacted with SsAtg1 to influence autophagic flux under starvation. Taken together, these results reveal that SsPtc3 responds to various stresses that modulate autophagy and phosphorylation of SsSmk1‐MAPK, which facilitates the growth and virulence of S. sclerotiorum.

Streptomyces scabies is a well‐researched plant pathogen belonging to the genus Streptomyces. Its virulence is linked to the production of the secondary metabolite thaxtomin A, which is tightly regulated at the transcriptional level. The leucine‐responsive regulatory protein (Lrp) family is conserved in prokaryotes and is involved in various crucial biological processes. However, the regulatory interaction between Lrp protein and pathogenic Streptomyces species remains poorly understood. This study aims to explore the role of SCAB_Lrp2 in regulating thaxtomin biosynthesis and pathogenicity, and to analyse the shared and unique features of Lrp homologues in S. scabies. We observed that SCAB_Lrp2 (SCAB_75421) showed significant homology with SCAB_Lrp, a recognised activator of thaxtomin A production in S. scabies. Our results revealed a regulatory interaction between SCAB_Lrp2 and SCAB_Lrp in terms of their targets, although SCAB_Lrp2 does not respond to the amino acid‐effectors of SCAB_Lrp. In contrast to SCAB_Lrp, deletion of SCAB_Lrp2 resulted in a notable increase in thaxtomin A production with the emergence of a hypervirulent phenotype in S. scabies. Further analysis revealed that SCAB_Lrp2 represses the transcription of the thaxtomin biosynthetic gene cluster by directly regulating the cluster‐situated regulator (CSR) gene txtR. Moreover, the precursor of thaxtomin, tryptophan, acts as an effector of SCAB_Lrp2, strengthening the repressive effect on thaxtomin biosynthesis through txtR. These findings provide new insights into the functional conservation and diversity of Lrp homologues involved in the biosynthesis of thaxtomin phytotoxins in pathogenic Streptomyces species.

Plant resistance, which acts as a selective pressure that affects viral population fitness, leads to the emergence of resistance‐breaking virus strains. Most recessive resistance to potyviruses is related to the mutation of eukaryotic translation initiation factor 4E (eIF4E) or its isoforms that break their interactions with the viral genome‐linked protein (VPg). In this study, we found that the VPg α1–α2 loop, which is essential for binding eIF4E, is the most variable domain of papaya ringspot virus (PRSV) VPg. PRSV VPg with the naturally occurring amino acid substitution of K105Q or E108G in the α1–α2 loop fails to interact with watermelon (Citrullus lanatus) eIF4E but interacts with watermelon eIF(iso)4E instead. Moreover, PRSV carrying these mutations can break the eIF4E‐mediated resistance to PRSV in watermelon accession PI 244019. We further revealed that watermelon eIF(iso)4E with the amino acid substitutions of DNQS to GAAA in the cap‐binding pocket could not interact with PRSV VPg with natural amino acid substitution of K105Q or E108G. Therefore, our finding provides a precise target for engineering watermelon germplasm resistant to resistance‐breaking PRSV isolates.

The production and scavenging of reactive oxygen species (ROS) are critical for plants to adapt to biotic and abiotic stresses. In this study, we investigated the interaction between the coat protein (CP) of cucumber green mottle mosaic virus (CGMMV) and ATP synthase subunit δ (ATPδ) in mitochondria. Silencing of ATPδ by tobacco rattle virus‐based virus‐induced gene silencing impeded CGMMV accumulation in Nicotiana benthamiana leaves. Both the overexpression of ATPδ in transgenic plants and transient expression promoted CGMMV infection. Nitro blue tetrazolium (NBT) and 3,3′‐diaminobenzidine (DAB) staining revealed that ATPδ inhibited O2⁻ production but not H2O2 production. The treatment of CGMMV‐infected leaves with the ROS inhibitor diphenylene iodonium (DPI) induced a ROS burst that inhibited CGMMV infection. Reverse transcription‐quantitative PCR and superoxide dismutase (SOD) activity assays showed that ATPδ, CGMMV infection, and CP expression specifically induced NbFeSOD3/4 expression and SOD activity, and silencing NbFeSOD3/4 inhibited CGMMV infection. We speculate that CGMMV CP interacts with ATPδ and hijacks it, thereby enhancing O2⁻ quenching by upregulating NbFeSOD expression and, in turn, SOD activity.

Obligate biotrophic powdery mildew fungi infect a wide range of economically important plants. These fungi often deliver effector proteins into the host tissues to suppress plant immunity and sustain infection. The phytohormone salicylic acid (SA) is one of the most important signals that activate plant immunity against pathogens. However, how powdery mildew effectors interact with host SA signalling is poorly understood. Isochorismatase (ISC) effectors from two other filamentous pathogens have been found to inhibit host SA biosynthesis by hydrolysing isochorismate, the main SA precursor in the plant cytosol. Here, we identified an ISC effector, named EqIsc1, from the rubber tree powdery mildew fungus Erysiphe quercicola. In ISC enzyme assays, EqIsc1 displayed ISC activity by transferring isochorismate to 2,3‐dihydro‐2,3‐dihydroxybenzoate in vitro and in transgenic Nicotiana benthamiana plants. In EqIsc1‐expressing transgenic Arabidopsis thaliana, SA biosynthesis and SA‐mediated immune response were significantly inhibited. In addition, we developed an electroporation‐mediated transformation method for the genetic manipulation of E. quercicola. Inoculation of rubber tree leaves with EqIsc1‐silenced E. quercicola strain induced SA‐mediated immunity. We also detected the translocation of EqIsc1 into the plant cytosol during the interaction between E. quercicola and its host. Taken together, our results suggest that a powdery mildew effector functions as an ISC enzyme to hydrolyse isochorismate in the host cytosol, altering the SA biosynthesis and immune response.

Jujube witches' broom, caused by phytoplasma, is a destructive disease of Chinese jujube. Studies have shown that zju‐miR156s play an important role in phytoplasma infection in jujube, but the regulatory mechanism between zju‐miR156c and witches' broom remains unexplored. In the current study, miRNA‐seq and gene expression analysis showed that zju‐miR156c was more highly induced in infected jujube plants than the other miRNAs and its target gene was ZjSPL3. In addition, the expression levels of thymidylate kinase gene (TMKJWB) and secreted jujube protein (SJP1JWB) in diseased materials were higher than those in healthy controls. The expression level of zju‐miR156c was significantly upregulated, while ZjSPL3 was sharply downregulated and the content of cytokinin (CTK) significantly increased. Overexpression of zju‐miR156c in Arabidopsis significantly reduced the expression of AtSPL10 (homologous gene of ZjSPL3) but increased the content of CTK, and the transgenic plants exhibited witches' broom symptoms. In addition, yeast two‐hybrid and co‐immunoprecipitation assays confirmed that SJP1JWB interacted with ZjERF18. Yeast one‐hybrid analysis showed that ZjERF18 could interact with the promoter of zju‐MIR156c. In conclusion, our results demonstrated a novel pathogenic module of ZjERF18‐zju‐miR156c‐ZjSPL3‐CTK has an important function in the formation of witches' broom caused by SJP1JWB.

Bacteria employ two‐component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum‐related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)‐related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein‐coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H2O2. Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxRD51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response.

The RaxX sulfopeptide, secreted via a type Ι secretion system, is crucial for activating XA21‐mediated innate immunity in resistant rice lines bearing the XA21 receptor kinase. Certain pathogenicity‐associated regulators that control the expression of the raxSTAB‐raxX gene cluster have been functionally characterized, but the comprehensive regulatory cascade of RaxSTAB and RaxX in Xanthomonas oryzae pv. oryzae (Xoo) remains incompletely understood. Our investigation revealed that pathogenicity‐associated regulators, including HrpG, HrpX, VemR, PhoR, and Clp, form a regulatory cascade governing the expression of the raxSTAB‐raxX gene cluster. HrpG regulates the raxSTAB‐raxX gene cluster transcription through the key regulator HrpX. VemR also participates in the transcription of the raxSTAB‐raxX. The histidine kinase PhoR positively modulates raxSTAB‐raxX expression, while the global regulator Clp directly binds the raxX promoter region to promote its transcription. These findings shed light on the intricate regulatory cascade of rax‐related genes in Xoo, emphasizing the complex roles of pathogenicity‐associated regulators within the pathogenic regulatory system.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases. The plant hormone abscisic acid (ABA) plays a key regulatory role in plant response to stress. ABA‐, stress‐, ripening‐induced proteins (ASR) have been shown to be abundantly induced in response to biotic and abiotic stresses to protect plants from damage. However, the function of wheat ASR2‐like protein (TaASR2L) in plants under biotic stress remains unclear. In this study, transient silencing of TaASR2L using a virus‐induced gene silencing system substantially reduced wheat resistance to Pst. TaASR2L interaction with serine/arginine‐rich splicing factor SR30‐like (TaSR30) was validated mainly in the nucleus. Knockdown of TaSR30 expression substantially reduced wheat resistance to Pst. Overexpression of TaASR2L and TaSR30 demonstrated that they can promote the expression of ABA‐ and resistance‐related genes to enhance wheat resistance to Pst. In addition, the expression levels of TaSR30 and TaASR2L were substantially increased by exogenous ABA, and the resistance of wheat to Pst was increased, and the expression of PR genes was induced. Therefore, these results suggest that TaASR2L interacts with TaSR30 by promoting PR genes expression and enhancing wheat resistance to Pst.

Plant diseases caused by fungal phytopathogens have led to significant economic losses in agriculture worldwide. The management of fungal diseases is mainly dependent on the application of fungicides, which are not suitable for sustainable agriculture, human health, and environmental safety. Thus, it is necessary to develop novel targets and green strategies to mitigate the losses caused by these pathogens. The target of rapamycin (TOR) complexes and key components of the TOR signalling pathway are evolutionally conserved in pathogens and closely related to the vegetative growth and pathogenicity. As indicated in recent systems, chemical, genetic, and genomic studies on the TOR signalling pathway, phytopathogens with TOR dysfunctions show severe growth defects and nonpathogenicity, which makes the TOR signalling pathway to be developed into an ideal candidate target for controlling plant disease. In this review, we comprehensively discuss the current knowledge on components of the TOR signalling pathway in microorganisms and the diverse roles of various plant TOR in response to plant pathogens. Furthermore, we analyse a range of disease management strategies that rely on the TOR signalling pathway, including genetic modification technologies and chemical controls. In the future, disease control strategies based on the TOR signalling network are expected to become a highly effective weapon for crop protection.

Potato virus Y (PVY, Potyviridae) is among the most important viral pathogens of potato. The potato resistance gene Nytbr confers hypersensitive resistance to the ordinary strain of PVY (PVYO), but not the necrotic strain (PVYN). Here, we unveil that residue 247 of PVY helper component proteinase (HCPro) acts as a central player controlling Nytbr strain‐specific activation. We found that substituting the serine at 247 in the HCPro of PVYO (HCProO) with an alanine as in PVYN HCPro (HCProN) disrupts Nytbr recognition. Conversely, an HCProN mutant carrying a serine at position 247 triggers defence. Moreover, we demonstrate that plant defences are induced against HCProO mutants with a phosphomimetic or another phosphorylatable residue at 247, but not with a phosphoablative residue, suggesting that phosphorylation could modulate Nytbr resistance. Extending beyond PVY, we establish that the same response elicited by the PVYO HCPro is also induced by HCPro proteins from other members of the Potyviridae family that have a serine at position 247, but not by those with an alanine. Together, our results provide further insights in the strain‐specific PVY resistance in potato and infer a broad‐spectrum detection mechanism of plant potyvirus effectors contingent on a single amino acid residue.

Ciboria shiraiana is a necrotrophic fungus that causes mulberry sclerotinia disease resulting in huge economic losses in agriculture. During infection, the fungus uses immunity elicitors to induce plant tissue necrosis that could facilitate its colonization on plants. However, the key elicitors and immune mechanisms remain unclear in C. shiraiana. Herein, a novel elicitor Cs08297 secreted by C. shiraiana was identified, and it was found to target the apoplast in plants to induce cell death. Cs08297 is a cysteine‐rich protein unique to C. shiraiana, and cysteine residues in Cs08297 were crucial for its ability to induce cell death. Cs08297 induced a series of defence responses in Nicotiana benthamiana, including the burst of reactive oxygen species (ROS), callose deposition, and activation of defence‐related genes. Cs08297 induced‐cell death was mediated by leucine‐rich repeat (LRR) receptor‐like kinases BAK1 and SOBIR1. Purified His‐tagged Cs08297‐thioredoxin fusion protein triggered cell death in different plants and enhanced plant resistance to diseases. Cs08297 was necessary for sclerotial development, oxidative‐stress adaptation, and cell wall integrity but negatively regulated virulence of C. shiraiana. In conclusion, our results revealed that Cs08297 is a novel fungal elicitor in fungi inducing plant immunity. Furthermore, its potential to enhance plant resistance provides a new target to control agricultural diseases biologically.

Plant root border cells (RBCs) prevent the colonization of plant growth‐promoting rhizobacteria (PGPR) at the root tip, rendering the PGPR unable to effectively control pathogens infecting the root tip. In this study, we engineered four strains of Pseudomonas sp. UW4, a typical PGPR strain, each carrying an enhanced green fluorescent protein (EGFP)‐expressing plasmid. The UW4E strain harboured only the plasmid, whereas the UW4E‐flg22 strain expressed a secreted EGFP‐Flg22 fusion protein, the UW4E‐Flg(flg22) strain expressed a non‐secreted Flg22, and the UW4E‐flg22‐D strain expressed a secreted Flg22‐DNase fusion protein. UW4E‐flg22 and UW4E‐flg22‐D, which secreted Flg22, induced an immune response in wheat RBCs and colonized wheat root tips, whereas the other strains, which did not secrete Flg22, failed to elicit this response and did not colonize wheat root tips. The immune response revealed that wheat RBCs synthesized mucilage, extracellular DNA, and reactive oxygen species. Furthermore, the Flg22‐secreting strains showed a 33.8%–93.8% higher colonization of wheat root tips and reduced the root rot incidence caused by Rhizoctonia solani and Fusarium pseudograminearum by 24.6%–35.7% compared to the non‐Flg22‐secreting strains in pot trials. There was a negative correlation between the incidence of wheat root rot and colonization of wheat root tips by these strains. In contrast, wheat root length and dry weight were positively correlated with the colonization of wheat root tips by these strains. These results demonstrate that engineered secretion of Flg22 by PGPR is an effective strategy for controlling root rot and improving plant growth.

Pathogens must efficiently acquire nutrients from host tissue to proliferate, and strategies to block pathogen access therefore hold promise for disease control. In this study, we investigated whether heme biosynthesis is an effective target for ablating the virulence of the phytopathogenic fungus Ustilago maydis on maize plants. We first constructed conditional heme auxotrophs of the fungus by placing the heme biosynthesis gene hem12 encoding uroporphyrinogen decarboxylase (Urod) under the control of nitrogen or carbon source‐regulated promoters. These strains were heme auxotrophs under non‐permissive conditions and unable to cause disease in maize seedlings, thus demonstrating the inability of the fungus to acquire sufficient heme from host tissue to support proliferation. Subsequent experiments characterized the role of endocytosis in heme uptake, the susceptibility of the fungus to heme toxicity as well as the transcriptional response to exogenous heme. The latter RNA‐seq experiments identified a candidate ABC transporter with a role in the response to heme and xenobiotics. Given the importance of heme biosynthesis for U. maydis pathogenesis, we tested the ability of the well‐characterized herbicide BroadStar to influence disease. This herbicide contains the active ingredient flumioxazin, an inhibitor of Hem14 in the heme biosynthesis pathway, and we found that it was an effective antifungal agent for blocking disease in maize. Thus, repurposing herbicides for which resistant plants are available may be an effective strategy to control pathogens and achieve crop protection.

Phytophthora infestans is a notorious oomycete pathogen that causes potato late blight. It secretes numerous effector proteins to manipulate host immunity. Understanding mechanisms underlying their host cell manipulation is crucial for developing disease resistance strategies. Here, we report that the conserved RXLR effector Pi05910 of P. infestans is a genotype‐specific avirulence elicitor on potato variety Longshu 12 and contributes virulence by suppressing and destabilizing host glycolate oxidase StGOX4. By performing co‐immunoprecipitation, yeast‐two‐hybrid assays, luciferase complementation imaging, bimolecular fluorescence complementation and isothermal titration calorimetry assays, we identified and confirmed potato StGOX4 as a target of Pi05910. Further analysis revealed that StGOX4 and its homologue NbGOX4 are positive immune regulators against P. infestans, as indicated by infection assays on potato and Nicotiana benthamiana overexpressing StGOX4 and TRV‐NbGOX4 plants. StGOX4‐mediated disease resistance involves enhanced reactive oxygen species accumulation and activated the salicylic acid signalling pathway. Pi05910 binding inhibited enzymatic activity and destabilized StGOX4. Furthermore, mutagenesis analyses indicated that the 25th residue (tyrosine, Y25) of StGOX4 mediates Pi05910 binding and is required for its immune function. Our results revealed that the core RXLR effector of P. infestans Pi05910 suppresses plant immunity by targeting StGOX4, which results in decreased enzymatic activity and protein accumulation, leading to enhanced plant susceptibility.

Persistent plant viruses are widespread in natural ecosystems. However, little is known about why persistent infection with these viruses may cause little or no harm to their host. Here, we discovered a new polerovirus that persistently infected wild rice plants by deep sequencing and assembly of virus‐derived small‐interfering RNAs (siRNAs). The new virus was named Rice tiller inhibition virus 2 (RTIV2) based on the symptoms developed in cultivated rice varieties following Agrobacterium‐mediated inoculation with an infectious RTIV2 clone. We showed that RTIV2 infection induced antiviral RNA interference (RNAi) in both the wild and cultivated rice plants as well as Nicotiana benthamiana. It is known that virulent virus infection in plants depends on effective suppression of antiviral RNAi by viral suppressors of RNAi (VSRs). Notably, the P0 protein of RTIV2 exhibited weak VSR activity and carries alanine substitutions of two amino acids broadly conserved among diverse poleroviruses. Mixed infection with umbraviruses enhanced RTIV2 accumulation and/or enabled its mechanical transmission in N. benthamiana. Moreover, replacing the alanine at either one or both positions of RTIV2 P0 enhanced the VSR activity in a co‐infiltration assay, and RTIV2 mutants carrying the corresponding substitutions replicated to significantly higher levels in both rice and N. benthamiana plants. Together, our findings show that as a persistent plant virus, RTIV2 carries specific mutations in its VSR gene to weaken viral suppression of antiviral RNAi. Our work reveals a new strategy for persistent viruses to maintain long‐term infection by weak suppression of the host defence response.

Vector‐borne bacterial pathogens cause devastating plant diseases that cost billions of dollars in crop losses worldwide. These pathogens have evolved to be host‐ and vector‐dependent, resulting in a reduced genome size compared to their free‐living relatives. All known vector‐borne bacterial plant pathogens belong to four different genera: ‘Candidatus Liberibacter’, ‘Candidatus Phytoplasma’, Spiroplasma and Xylella. To protect themselves against pathogens, plants have evolved pattern recognition receptors that can detect conserved pathogen features as non‐self and mount an immune response. To gain an understanding of how vector‐borne pathogen features are perceived in plants, we investigated three proteinaceous features derived from cold shock protein (csp22), flagellin (flg22) and elongation factor Tu (elf18) from vector‐borne bacterial pathogens as well as their closest free‐living relatives. In general, vector‐borne pathogens have fewer copies of genes encoding flagellin and cold shock protein compared to their closest free‐living relatives. Furthermore, epitopes from vector‐borne pathogens were less likely to be immunogenic compared to their free‐living counterparts. Most Liberibacter csp22 and elf18 epitopes do not trigger plant immune responses in tomato or Arabidopsis. Interestingly, csp22 from the citrus pathogen ‘Candidatus Liberibacter asiaticus’ triggers immune responses in solanaceous plants, while csp22 from the solanaceous pathogen ‘Candidatus Liberibacter solanacearum’ does not. Our findings suggest that vector‐borne plant pathogenic bacteria evolved to evade host recognition.

Following the invasion by the pine wood nematode (PWN) into north‐east China, a notable disparity in susceptibility was observed among Pinaceae species. Larix olgensis exhibited marked resilience and suffered minimal fatalities, while Pinus koraiensis experienced significant mortality due to PWN infection. Our research demonstrated that the PWNs in L. olgensis showed a 13.43% reduction in lipid content compared to P. koraiensis (p < 0.05), which was attributable to the accumulation of caffeic acid in L. olgensis. This reduction in lipid content was correlated with a decreased overwintering survival of PWNs. The diminished lipid reserves were associated with substantial stunting in PWNs, including reduced body length and maximum body width. The result suggests that lower lipid content is a major factor contributing to the lower overwintering survival rate of PWNs in L. olgensis induced by caffeic acid. Through verification tests, we concluded that the minimal fatalities observed in L. olgensis could be attributed to the reduced overwintering survival of PWNs, a consequence of caffeic acid‐induced stunting. This study provides valuable insights into PWN–host interactions and suggests that targeting caffeic acid biosynthesis pathways could be a potential strategy for managing PWN in forest ecosystems.

Pear chlorotic leaf spot‐associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single‐stranded, negative‐sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens‐mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self‐interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell‐to‐cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N‐terminal 1–89 amino acids of the P5 were responsible for the self‐interaction ability and RSS activity, for which the signal peptide at positions 1–19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.

Positive‐sense RNA viruses remodel cellular cytoplasmic membranes as the membranous sources for the formation of viral replication organelles (VROs) for viral genome replication. In plants, they traffic through plasmodesmata (PD), plasma membrane‐lined pores enabling cytoplasmic connections between cells for intercellular movement and systemic infection. In this study, we employed turnip mosaic virus (TuMV), a plant RNA virus to investigate the involvement of RTNLB3 and RTNLB6, two ER (endoplasmic reticulum) membrane‐bending, PD‐located reticulon‐like (RTNL) non‐metazoan group B proteins (RTNLBs) in viral infection. We show that RTNLB3 interacts with TuMV 6K2 integral membrane protein and RTNLB6 binds to TuMV coat protein (CP). Knockdown of RTNLB3 promoted viral infection, whereas downregulation of RTNLB6 restricted viral infection, suggesting that these two RTNLs play contrasting roles in TuMV infection. We further demonstrate that RTNLB3 targets the α‐helix motif ⁴²LRKSM⁴⁶ of 6K2 to interrupt 6K2 self‐interactions and compromise 6K2‐induced VRO formation. Moreover, overexpression of AtRTNLB3 apparently promoted the selective degradation of the ER and ER‐associated protein calnexin, but not 6K2. Intriguingly, mutation of the α‐helix motif of 6K2 that is required for induction of VROs severely affected 6K2 stability and abolished TuMV infection. Thus, RTNLB3 attenuates TuMV replication, probably through the suppression of 6K2 function. We also show that RTNLB6 promotes viral intercellular movement but does not affect viral replication. Therefore, the proviral role of RTNLB6 is probably by enhancing viral cell‐to‐cell trafficking. Taken together, our data demonstrate that RTNL family proteins may play diverse complex, even opposite, roles in viral infection in plants.

MicroRNA‐like RNAs (milRNAs) play a significant role in the infection process by plant‐pathogenic fungi. However, the specific functions and regulatory mechanisms of fungal milRNAs remain insufficiently elucidated. This study investigated the function of Foc‐milR138, an infection‐induced milRNA secreted by Fusarium oxysporum f. sp. cubense (Foc), which is the causal agent of Fusarium wilt of banana. Initially, through precursor gene knockout and phenotypic assessments, we confirmed that Foc‐milR138 acts as a virulent milRNA prominently upregulated during the early stages of Foc infection. Subsequent bioinformatic analyses and transient expression assays in Nicotiana benthamiana leaves identified a host receptor‐like kinase gene, MaLYK3, as the direct target of Foc‐milR138. Functional investigations of MaLYK3 revealed its pivotal role in triggering immune responses of N. benthamiana by upregulating a suite of resistance genes, bolstering reactive oxygen species (ROS) accumulation and callose deposition, thereby fortifying disease resistance. This response was markedly subdued upon co‐expression with Foc‐milR138. Expression pattern analysis further verified the specific suppression of MaLYK3 by Foc‐milR138 during the early root infection by Foc. In conclusion, Foc secretes a virulent milRNA (Foc‐milR138) to enter the host banana cells and inhibit the expression of the plant surface receptor‐like kinase MaLYK3, subverting the disease resistance activated by MaLYK3, and ultimately facilitating pathogen invasion. These findings shed light on the roles of fungal milRNAs and their targets in resistance and pathogenicity, offering promising avenues for the development of disease‐resistant banana cultivars.

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI)‐suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors' ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI‐suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI‐suppressing effectors. From among the libraries, multiple members of the killer protein‐like 4 (KP4) and killer protein‐like 6 (KP6) effector families were identified as PTI suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection and suggests that, similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonization.

ATP‐binding cassette (ABC) transporters hydrolyse ATP to transport various substrates. Previous studies have shown that ABC transporters are responsible for transporting plant hormones and heavy metals, thus contributing to plant immunity. Herein, we identified a wheat G‐type ABC transporter, TaABCG2‐5B, that responds to salicylic acid (SA) treatment and is induced by Fusarium graminearum, the primary pathogen causing Fusarium head blight (FHB). The loss‐of‐function mutation of TaABCG2‐5B (ΔTaabcg2‐5B) reduced SA accumulation and increased susceptibility to F. graminearum. Conversely, overexpression of TaABCG2‐5B (OE‐TaABCG2‐5B) exerted the opposite effect. Quantification of intracellular SA in ΔTaabcg2‐5B and OE‐TaABCG2‐5B protoplasts revealed that TaABCG2‐5B acts as an importer, facilitating the transport of SA into the cytoplasm. This role was further confirmed by Cd²⁺ absorption experiments in wheat roots, indicating that TaABCG2‐5B also participates in Cd²⁺ transport. Thus, TaABCG2‐5B acts as an importer and is crucial for transporting multiple substrates. Notably, the homologous gene TaABCG2‐5A also facilitated Cd²⁺ uptake in wheat roots but did not significantly influence SA accumulation or FHB resistance. Therefore, TaABCG2 could be a valuable target for enhancing wheat tolerance to Cd²⁺ and improving FHB resistance.

The myo‐inositol‐1‐phosphate synthase (MIPS) catalyses the biosynthesis of myo‐inositol, an important sugar that regulates various physiological and biochemical processes in plants. Here, we provide evidence that host (SlMIPS1) and pathogen (Rs_MIPS) myo‐inositol‐1‐phosphate synthase (MIPS) genes are required for successful infection of Rhizoctonia solani, a devastating necrotrophic fungal pathogen, in tomato. Silencing of either SlMIPS1 or Rs_MIPS prevented disease, whereas an exogenous spray of myo‐inositol enhanced disease severity. SlMIPS1 was upregulated upon R. solani infection, and potentially promoted source‐to‐sink transition, induced SWEET gene expression, and facilitated sugar availability in the infected tissues. In addition, salicylic acid (SA)‐jasmonic acid homeostasis was altered and SA‐mediated defence was suppressed; therefore, disease was promoted. On the other hand, silencing of SlMIPS1 limited sugar availability and induced SA‐mediated defence to prevent R. solani infection. Virus‐induced gene silencing of NPR1, a key gene in SA signalling, rendered SlMIPS1‐silenced tomato lines susceptible to infection. These analyses suggest that induction of SA‐mediated defence imparts disease tolerance in SlMIPS1‐silenced tomato lines. In addition, we present evidence that SlMIPS1 and SA negatively regulate each other to modulate the defence response. SA treatment reduced SlMIPS1 expression and myo‐inositol content in tomato, whereas myo‐inositol treatment prevented SA‐mediated defence. We emphasize that downregulation of host/pathogen MIPS can be an important strategy for controlling diseases caused by R. solani in agriculturally important crops.

Understanding how pathogens defend themselves against host defence mechanisms, such as hydrogen peroxide (H2O2) production, is crucial for comprehending fungal infections. H2O2 poses a significant threat to invading fungi due to its potent oxidizing properties. Our research focuses on the hemibiotrophic fungal wheat pathogen Zymoseptoria tritici, enabling us to investigate host–pathogen interactions. We examined two catalase‐peroxidase (CP) genes, ZtCpx1 and ZtCpx2, to elucidate how Z. tritici deals with host‐generated H2O2 during infection. Our analysis revealed that ZtCpx1 was up‐regulated during biotrophic growth and asexual spore formation in vitro, while ZtCpx2 showed increased expression during the transition from biotrophic to necrotrophic growth and in‐vitro vegetative growth. Deleting ZtCpx1 increased the mutant's sensitivity to exogenously added H2O2 and significantly reduced virulence, as evidenced by decreased Septoria tritici blotch symptom severity and fungal biomass production. Reintroducing the wild‐type ZtCpx1 allele with its native promoter into the mutant strain restored the observed phenotypes. While ZtCpx2 was not essential for full virulence, the ZtCpx2 mutants exhibited reduced fungal biomass development during the transition from biotrophic to necrotrophic growth. Moreover, both CP genes act synergistically, as the double knock‐out mutant displayed a more pronounced reduced virulence compared to ΔZtCpx1. Microscopic analysis using fluorescent proteins revealed that ZtCpx1 was localized in the peroxisome, indicating its potential role in managing host‐generated reactive oxygen species during infection. In conclusion, our research sheds light on the crucial roles of CP genes ZtCpx1 and ZtCpx2 in the defence mechanism of Z. tritici against host‐generated hydrogen peroxide.