Histological And Cytological Analyses Of Adult Plant Resistance To Wheat Stripe Rust And Characterization Of Non-host Resistance Of Wheat To Uromyces Fabae

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the mostdamaging diseases of wheat in China. Adult plant resistance (APR) to stripe rust is knownto be an important component of some sources of durable resistance in wheat, thus has greatpractical potentials in wheat production. Characterization of APR to stripe rust at histologicaland cytological level will set the foundation for further elucidation the mechanism of APR atmolecular level, therfore, will be of greatly important for appropriate utilization of resistantcultivars and sustainable control of the disease. Thus the present study was conducted toelucidate the phenotype of XZ9104(XZ) relative to leaf position and different plant growthstages by Pst(CYR32) inoculation. In addition, histopathological analysis was undertaken tocharacterize the cellular interaction between Pst and XZ at seedling (GS12), tillering(GS21-22) and boot stages (GS43-49), which represent the expression of resistance transitionfrom susceptibility to resistance. Also, the accumulation of ROS (reactive oxygen species)and variation in resistance in this interaction were studied systematically using histochemicalmethods. Finally, transmission electron microscopy (TEM) was used to describeultrastructural changes during XZ and Pst interactions. Our study will character the APR inXZ to Pst at histological and cytological level. The main results are as follows:1. To elucidate the correlation of APR expression and plant development, the leaves invarious positions at different growth stages were inoculated simultaneously with Pst raceCYR32in the greenhouse. The study result indicated that APR was initiated as early astillering stage, increased gradually as plants grew, and most effective at the boot stage.2. The microscopical observation revealed that the time points of the appearance ofhaustorium formation in the infection sites of plant leaves in the seedling, tillering and bootstages were12,18and24hai, respectively, although the pathogen entered plant leaves andformed a substomatal vesicle at12hai in all stages. It indicated that haustorial formation wasretarded or inhibited in adult plants. The hypha branching and length of hypha as well asnumber of haustorial mother cell (HMC) in all growth stages were similar until48hai andwithout obvious difference between all growth stages. In the seedling-stage plants, after48hai, massive formation of secondary hyphae as Pst proliferated rapidly which subsequently confer the formation of small microcolony. In most of infection site, the secondary hyphaappeared at96hai in boot-stage plants. It indicated that the differentiation of secondaryhyphae was significantly inhibited, which decreased the development of microcolonies inplants of boot stage. One of the observable responses at the microscopic level was thenecrosis associated with the invaded plant cells coincided with the haustorial formation in thetillering and boot stages plants. The typical hypersensitive response (HR) was observed from18to36hai in leaves of plants in tillering and boot stages, respectively. With a longerincubation time, the percentage of infection sites with necrotic host cells and the necrotic areaincreased in inoculated leaves of plants in both tillering and boot stages, necrotic host cellswere observed in80%of the infection sites in the tillering plants by120hai and in100%ofthe infection sites of the boot-stage plants at144hai. At some infection site of adult plant,the infection unit were completely surrounded by the necrotic tissue which render the fungalabortion and lack of secondary hypha formation. It indicated that the death of host cellsmarkedly inhibited the development of hyphae.3. The result of histochemical location of ROS revealed that a high frequency of NBT(Nitroblue tetrazolium) stained cells showed O₂accumulation was detected at24hai ontillering stage plants and at36hai on boot-stage plants, respectively. The high frequency ofNBT stained cells coincided with the infection stage when contacts were established betweenthe HMCs and the mesophyll cells and haustoria began to form, and while the host cells withNBT staining remained alive. It indicated that the production of O₂preceded HR of infectedcells. The H₂O₂accumulations in infected leaves of plants in tillering and booting stages weresimilar and both showed a biphasic process. On tillering plants, The first and second peak ofH₂O₂generation in invaded host cells appeared at24hai and72-96hai, respectively. Whileon boot plants these peaks appeared at48hai and120hai, respectively. The first burst phaseof H₂O₂coincided with the beginning of haustorium formation and the subsequent HR ininvaded host cells, while the second phase of H₂O₂generation coincided with the formation ofsecondary hyphae and the increasing number of necrotic host cells surrounding the attackedcells. In the infected leaves of seedlings, in contrast, at most infection sites, the pathogeninfection didn't induce obvious H₂O₂and O₂generation. The result suggested that, thegeneration and accumulation of O₂-and H₂O₂are closely associated with the HR of host cells,and which may play an important role in the expression of APR.4. The result of TEM study revealed that the typical APR reactions of XZ at the subcellularlevel included significant cell wall-related resistance, necrosis of penetrated host cells, and theformation of a haustorial sheath. These structural defense responses are believed tosignificantly inhibit the growth of Pst by accelerating the senescence of hyphae and cell death of infection structures.Non-host resistance (NHR), which is defined as resistance shown by all genotypes of aplant species against all genotypes of a given pathogen species, is the most common form ofdisease resistance exhibited by plants against the majority of potentially pathogenicmicroorganisms. NHR to cereal rusts has proved to be durable and appears to have greatpotential for engineering plant immunity. Little is known about the nature of this effectivedefense mechanism to the pathogen of remotely related plant species in important cropspecies, wheat, which can be damaged by several Puccinia species, P. striiformis f. sp. tritici,P. triticina and P. graminis f. sp. tritici, but is immune to all Uromyces species. Here weestablished a novel non-adapted pathosystem based on wheat (Triticum aestivum) and thebroad bean rust pathogen Uromyces fabae (Uf). We first investigated the reaction of a wheatcultivar to the infection by the broad bean rust pathogen using light and electron microscopes.Histochemical experiments were conducted to detect ROS generation in the non-hostpathosystem. Expression profiles of candidate genes related to basal resistance and oxidativestress in wheat leaves inoculated with Uf were assayed by quantitative RT-PCR (qRT-PCR).Our study will revealed the cytological and molecular basis of NHR in wheat against rustpathogen of remotely related plant species and lead to a comprehensive understanding ofresistance in wheat against rust fungi.1. In the wheat-Uf interaction, microscopic observations showed that urediospores germinatedefficiently on wheat leaves. However, over98%of the germ tubes failed to form appressoriaover stomata and developed substomatal vesicles inside wheat leaves. Therefore, stomapenetration was significantly reduced in Uf infection of wheat. For the few that invadedthrough stomata and formed the intercellular hypha and haustorial mother cell, the majority ofHMC failed to penetrate wheat mesophyll cells. At96hours after inoculation, less than4%ofthe Uf infection units that had entered the mesophyll tissue formed haustoria.2. The result of TEM study revealed that the Attempted penetration by haustorium mothercells induced the thickening of cell wall and the formation of papillae in plant cells, thepenetration peg emerging from the HMC appeared to puncture through the plant cell wall butfailed to penetrate through the papilla and form haustoria in wheat cells. Thus, it indicatedthat cell wall-related resistance arrested the development or growth of Uf penetration pegs andNHR of wheat to Uf is pre-haustorial. Although a few penetration pegs could breach the cellwall and periphery deposits and formed haustoria, the light microscopic observation revealedthat these haustoria formed by Uf in wheat cells were encased in callose-like deposits that hadstrong auto-fluorescence. Moreover, the formation of haustoria did not trigger thehypersensitive response in penetrated wheat cells, which was consistent with the macroscopic phenotype.2. The result of histochemical location of AOS revealed that in responses to Uf infection,accumulation of H₂O₂, but not O₂, was detected in wheat cells. H₂O₂was mainly detected incell wall in close contact with IH or HMCs, papillae subjacent to HMC, and depositsresponsible for haustorium encasement. The cross-link fueled by H₂O₂likely reinforces thecell wall at the sites in contact with fungal structures and strengthening papillae as well as theencasement of haustoria and results in the arrest of Uf growth in wheat.3. The putative TaPR1(pathogenesis-related protein1), TaPR2(-1,3-glucanase), TaPR3(chitinase), TaPR5(thaumatin-like), TaPAL (phenylalanine ammonia lyase), TaSOD(superoxide dismutase), TaCAT (catalase), and TaAPX (ascorbate-peroxidase) genes wereselected as candidate defense-related genes for assaying their expression levels during Ufinfection by qRT-PCR. Although they had different expression profiles, all of these eightgenes were up-regulated at one or more time-points between12-48hai. It revealed that thesedefense related genes also may contribute to preventing Uf infection of wheat, genes involvedin oxidative stress may regulate the ROS generation and avoid over-accumulation of ROS andeliciting HR, similar PR genes have been implicated in wheat resistance against both adaptedand non-adapted rust fungi may indicate that NHR and basal resistance to adapted pathogensmay involve the same or similar mechanisms in wheat.